Compositions and methods for inhibiting nuclear receptor subfamily 1 group h member 3 (nr1h3) expression

ABSTRACT

Oligonucleotides are provided herein that inhibit NR1H3 expression. Also provided are compositions including the same and uses thereof, particularly uses relating to treating diseases, disorders and/or conditions associated with NR1H3 expression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/724,176, filed Apr. 19, 2022, which claims priority to EuropeanPatent Applications 21213711.1, filed Dec. 10, 2021 and 21186366.7,filed Jul. 19, 2021, and claims priority to U.S. Provisional PatentApplication 63/176,814, filed Apr. 19, 2021; the contents of which areincorporated herein by reference.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in XML format via the USPTO patent electronic filing systemand is hereby incorporated by reference in its entirety. Said XML file,created on Jan. 3, 2023, is named 210010US03.xml and is 4770 kilobytesin size.

BACKGROUND

The liver plays a critical role in the metabolism of lipids.Abnormalities in normal hepatic lipid metabolism are associated with thedevelopment of various liver diseases or disorders such as non-alcoholicfatty liver disease (NAFLD) and its subsequent progression tonon-alcoholic steatohepatitis (NASH) and potentially other advancedliver abnormalities.

NAFLD is one of the most common liver diseases, with increasingprevalence worldwide (Loomba R., & Sanyal A. J. NAT. REV. GASTROENTEROLHEPATOL. (2013); 10(11): 686-90). NAFLD is a liver disease characterizedby a spectrum of clinical and pathological severity ranging from simplesteatosis to nonalcoholic fatty liver (NAFL), nonalcoholicsteatohepatitis (NASH), fibrosis, cirrhosis, hepatocellular carcinoma(HCC) and liver failure (Bessone F, et al., CELL MOL. LIFE SCI. (2019);76(1): 99-128). NAFLD is characterized by the presence of fat in theliver in the absence of significant alcohol use and other causes of fataccumulation in the liver such as medications, starvation, and viraldisease (Chalasani, N., et al., HEPATOLOGY (Baltimore, Md.), (2012);55(6): 2005-23). Additionally, as the disease progresses, NASH patientsalso have an increased risk of developing extra-hepatic complications,particularly cardiovascular diseases (CVD), which are among the mostcommon causes of death in this patient population (Patil R, et al.,WORLD J. GASTROINTEST. PATHOPHYSIOL. (2017); 8(2): 51-8). Theabnormalities in hepatic lipid metabolism that lead to NAFLD also drivethe progression of atherogenic dyslipidemia, where elevated plasmatriglycerides (TG), cholesterol and lipoprotein particles infiltrate thearterial wall and subsequently develop atherosclerotic plaques (Loomba R& Sanyal A J NAT. REV. GASTROENTEROL. HEPATOL. (2013); 10(11): 686-90).Thus, there is a significant medical need for the development of diseasemodifying therapeutics for NAFLD.

SUMMARY OF DISCLOSURE

The disclosure is based in part on the discovery of oligonucleotides(e.g., RNAi oligonucleotides) that reduce NR1H3 (nuclear receptorsubfamily 1, group H, member 3) expression in the liver. Specifically,target sequences within NR1H3 mRNA were identified and oligonucleotidesthat bind to these target sequences and inhibit NR1H3 mRNA expressionwere generated. As demonstrated herein, the oligonucleotides inhibitedmurine, monkey and/or human NR1H3 expression in the liver. Without beingbound by theory, the oligonucleotides described herein are useful fortreating a disease, disorder or condition associated with NR1H3expression (e.g., non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), or systemic lupus erythematosus).

In an aspect, the invention provides an RNAi oligonucleotide forreducing NR1H3 expression, the oligonucleotide comprising a sense strandand an antisense strand, wherein the sense strand and the antisensestrand form a duplex region, wherein the antisense strand comprises aregion of complementarity to a NR1H3 mRNA target sequence of any one ofSEQ ID NOs: 1-384, and wherein the region of complementarity is at least15 contiguous nucleotides in length. In an aspect, the inventionprovides an RNAi oligonucleotide for reducing NR1H3 expression, theoligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein the antisense strand comprises a region of complementarity to aNR1H3 mRNA target sequence of any one of SEQ ID NOs: 1125-1511, andwherein the region of complementarity is at least 15 contiguousnucleotides in length.

In some embodiments, the RNAi oligonucleotide comprises (i) an antisensestrand of 19-30 nucleotides in length, wherein the antisense strandcomprises a nucleotide sequence comprising a region of complementarityto a NR1H3 mRNA target sequence, wherein the region of complementarityis selected from SEQ ID NOs: 786, 787, 1537 and 813, and (ii) a sensestrand of 19-50 nucleotides in length comprising a region ofcomplementarity to the antisense strand, wherein the antisense and sensestrands are separate strands which form an asymmetric duplex regionhaving an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments, the RNAi oligonucleotide comprises (i) an antisensestrand of 19-30 nucleotides in length, wherein the antisense strandcomprises a nucleotide sequence comprising a region of complementarityto a NR1H3 mRNA target sequence, wherein the region of complementarityis selected from SEQ ID NOs: 1509, 1510 1409, and 1511 and (ii) a sensestrand of 19-50 nucleotides in length comprising a region ofcomplementarity to the antisense strand, wherein the antisense and sensestrands are separate strands which form an asymmetric duplex regionhaving an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments of the RNAi oligonucleotide: (i) the sense strand is15 to 50 or 18 to 36 nucleotides in length, optionally 36 nucleotides inlength; optionally (ii) the antisense strand is 15 to 30 nucleotides inlength, optionally 22 nucleotides in length; and optionally (iii) theduplex region is at least 19 nucleotides or at least 20 nucleotides inlength.

In some embodiments of the RNAi oligonucleotide, the region ofcomplementarity is at least 19 contiguous nucleotides in length,optionally at least 20 contiguous nucleotides in length, optionally 20contiguous nucleotides, and optionally wherein the region ofcomplementarity is fully complementary to the mRNA target sequence atnucleotide positions 2-8 of the antisense strand or positions 2-11 ofthe antisense strand, nucleotide numbering 5′ to 3′.

In some embodiments of the RNAi oligonucleotide: the 3′ end of the sensestrand comprises a stem-loop set forth as S1-L-S2, wherein (i) S1 iscomplementary to S2, optionally wherein S1 and S2 are each 1-10nucleotides in length and have the same length, optionally wherein S1and S2 are each 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length,further optionally wherein S1 and S2 are 6 nucleotides in length; and(ii) L forms a loop between S1 and S2 of 3-5 nucleotides in length,optionally wherein L is a triloop or a tetraloop, optionally wherein thetetraloop comprises the sequence 5′-GAAA-3′, optionally wherein thestem-loop comprises the sequence 5′-GCAGCCGAAAGGCUGC-3′ (SEQ ID NO:1121). In some embodiments of the RNAi oligonucleotide comprises anicked tetraloop structure or comprises a nick between the 3′ terminusof the sense strand and the 5′ terminus of the antisense strand.

In some embodiments of the RNAi oligonucleotide: the antisense strandcomprises an overhang sequence of one or more nucleotides in length atthe 3′ terminus, optionally wherein the overhang comprises purinenucleotides, optionally wherein the overhang sequence is 2 nucleotidesin length, optionally wherein the overhang is selected from AA, GG, AG,and GA, optionally wherein the overhang is GG.

In some embodiments of the RNAi oligonucleotide: (i) the oligonucleotidecomprises at least one modified nucleotide, optionally wherein themodified nucleotide comprises a 2′-modification, optionally wherein: (a)the 2′-modification is a modification selected from 2′-aminoethyl,2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid, optionally wherein themodification is selected from 2′-fluoro and 2′-O-methyl, optionallywherein all nucleotides of the oligonucleotide are modified, wherein themodification is 2′-fluoro and 2′-O-methyl; (b) about 10-15%, 10%, 11%,12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a2′-fluoro modification; (c) about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strandcomprise a 2′-fluoro modification; (d) about 25-35%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of theoligonucleotide comprise a 2′-fluoro modification; (e) the sense strandcomprises 36 nucleotides with positions 1-36 from 5′ to 3′, whereinpositions 8-11 comprise a 2′-fluoro modification; (f) the antisensestrand comprises 22 nucleotides with positions 1-22 from 5′ to 3′, andwherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2′-fluoromodification; and/or (g) the remaining nucleotides comprise a2′-O-methyl modification, and/or (ii) the oligonucleotide comprises atleast one modified internucleotide linkage, optionally wherein the atleast one modified internucleotide linkage is a phosphorothioatelinkage, optionally wherein: (a) the antisense strand comprises aphosphorothioate linkage (i) between positions 1 and 2, and betweenpositions 2 and 3; or (ii) between positions 1 and 2, between positions2 and 3, and between positions 3 and 4, wherein positions are numbered1-4 from 5′ to 3′; and/or (b) the antisense strand is 22 nucleotides inlength, and wherein the antisense strand comprises a phosphorothioatelinkage between positions 20 and 21 and between positions 21 and 22,wherein positions are numbered 1-22 from 5′ to 3′, and/or (iii) theantisense strand comprises a phosphorylated nucleotide at the 5′terminus, wherein the phosphorylated nucleotide is selected from uridineand adenosine, optionally wherein the phosphorylated nucleotide isuridine, and/or (iv) the 4′-carbon of the sugar of the 5′-nucleotide ofthe antisense strand comprises a phosphate analog, optionally whereinthe phosphate analog is oxymethylphosphonate, vinylphosphonate ormalonylphosphonate, optionally wherein the phosphate analog is a4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy, and/or (v)at least one nucleotide of the oligonucleotide is conjugated to one ormore targeting ligands, optionally wherein: (a) each targeting ligandcomprises a carbohydrate, amino sugar, cholesterol, polypeptide orlipid; (b) the stem loop comprises one or more targeting ligandsconjugated to one or more nucleotides of the stem loop; (c) the one ormore targeting ligands is conjugated to one or more nucleotides of theloop, optionally wherein the loop comprises 4 nucleotides numbered 1-4from 5′ to 3′, wherein nucleotides at positions 2, 3, and 4 eachcomprise one or more targeting ligands, wherein the targeting ligandsare the same or different; (d) each targeting ligand comprises aN-acetylgalactosamine (GalNAc) moiety, optionally wherein the GalNAcmoiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, atrivalent GalNAc moiety or a tetravalent GalNAc moiety; and/or (e) up to4 nucleotides of L of the stem-loop are each conjugated to a monovalentGalNAc moiety.

In some embodiments of the RNAi oligonucleotide, the targeting ligandcomprises at least one GalNAc moiety and targets human liver cells(e.g., human hepatocytes).

In some embodiments of the RNAi oligonucleotide: (i) the sense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 769-856, SEQID NOs: 1519-1552, SEQ ID NOs: 1409, 1509-1511, or SEQ ID NOs: 945-1032,optionally a nucleotide sequence selected from SEQ ID NOs: SEQ ID NOs:786, 787, 813, and 1537; and optionally (ii) the antisense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 857-944 or SEQID NOs: 1033-1120, optionally a nucleotide sequence selected from SEQ IDNOs: 874, 875, 901, and 929.

In some embodiments of the RNAi oligonucleotide: the sense strand andantisense strands comprise nucleotide sequences selected from the groupconsisting of:(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively.

In some embodiments of the RNAi oligonucleotide: the sense and antisensestrands comprise nucleotide sequences selected from the group consistingof:

(a) SEQ ID NOs: 945 and 1033, respectively;(b) SEQ ID NOs: 946 and 1034, respectively;(c) SEQ ID NOs: 947 and 1035, respectively;(d) SEQ ID NOs: 948 and 1036, respectively;(e) SEQ ID NOs: 949 and 1037, respectively;(f) SEQ ID NOs: 950 and 1038, respectively;(g) SEQ ID NOs: 951 and 1039, respectively;(h) SEQ ID NOs: 952 and 1040, respectively;(i) SEQ ID NOs: 953 and 1041, respectively;(j) SEQ ID NOs: 954 and 1042, respectively;(k) SEQ ID NOs: 955 and 1043, respectively;(l) SEQ ID NOs: 956 and 1044 respectively;(m) SEQ ID NOs: 957 and 1045, respectively;(n) SEQ ID NOs: 958 and 1046, respectively;(o) SEQ ID NOs: 959 and 1047, respectively;(p) SEQ ID NOs: 960 and 1048, respectively;(q) SEQ ID NOs: 961 and 1049, respectively;(r) SEQ ID NOs: 962 and 1050, respectively;(s) SEQ ID NOs: 963 and 1051, respectively;(t) SEQ ID NOs: 964 and 1052, respectively;(u) SEQ ID NOs: 965 and 1053, respectively;(v) SEQ ID NOs: 966 and 1054, respectively;(w) SEQ ID NOs: 967 and 1055, respectively;(x) SEQ ID NOs: 968 and 1056, respectively;(y) SEQ ID NOs: 969 and 1057, respectively;(z) SEQ ID NOs: 970 and 1058, respectively;(aa) SEQ ID NOs: 971 and 1059, respectively;(bb) SEQ ID NOs: 972 and 1060, respectively;(cc) SEQ ID NOs: 973 and 1061, respectively;(dd) SEQ ID NOs: 974 and 1062, respectively;(ee) SEQ ID NOs: 975 and 1063, respectively;(ff) SEQ ID NOs: 976 and 1064, respectively;(gg) SEQ ID NOs: 977 and 1065, respectively;(hh) SEQ ID NOs: 978 and 1066, respectively;(ii) SEQ ID NOs: 979 and 1067, respectively;(jj) SEQ ID NOs: 980 and 1068, respectively;(kk) SEQ ID NOs: 981 and 1069, respectively;(ll) SEQ ID NOs: 982 and 1070, respectively;(mm) SEQ ID NOs: 983 and 1071, respectively;(nn) SEQ ID NOs: 984 and 1072, respectively;(oo) SEQ ID NOs: 985 and 1073, respectively;(pp) SEQ ID NOs: 986 and 1074, respectively;(qq) SEQ ID NOs: 987 and 1075, respectively;(rr) SEQ ID NOs: 988 and 1076, respectively;(ss) SEQ ID NOs: 989 and 1077, respectively;(tt) SEQ ID NOs: 990 and 1078, respectively;(uu) SEQ ID NOs: 991 and 1079, respectively;(vv) SEQ ID NOs: 992 and 1080, respectively;(ww) SEQ ID NOs: 993 and 1081, respectively;(xx) SEQ ID NOs: 994 and 1082, respectively;(yy) SEQ ID NOs: 995 and 1083, respectively;(zz) SEQ ID NOs: 996 and 1084, respectively;(aaa) SEQ ID NOs: 997 and 1085, respectively;(bbb) SEQ ID NOs: 998 and 1086, respectively;(ccc) SEQ ID NOs: 999 and 1087, respectively;(ddd) SEQ ID NOs: 1000 and 1088, respectively;(eee) SEQ ID NOs: 1001 and 1089, respectively;(fff) SEQ ID NOs: 1002 and 1090, respectively;(ggg) SEQ ID NOs: 1003 and 1091, respectively;(hhh) SEQ ID NOs: 1004 and 1092 respectively;(iii) SEQ ID NOs: 1005 and 1093 respectively;(jjj) SEQ ID NOs: 1006 and 1094, respectively;(kkk) SEQ ID NOs: 1007 and 1095, respectively;(lll) SEQ ID NOs: 1008 and 1096, respectively;(mmm) SEQ ID NOs: 1009 and 1097, respectively;(nnn) SEQ ID NOs: 1010 and 1098, respectively;(ooo) SEQ ID NOs: 1011 and 1099, respectively;(ppp) SEQ ID NOs: 1012 and 1100, respectively;(qqq) SEQ ID NOs: 1013 and 1101, respectively;(rrr) SEQ ID NOs: 1014 and 1102 respectively;(sss) SEQ ID NOs: 1015 and 1103, respectively;(ttt) SEQ ID NOs: 1016 and 1104, respectively;(uuu) SEQ ID NOs: 1017 and 1105, respectively;(vvv) SEQ ID NOs: 1018 and 1106, respectively;(www) SEQ ID NOs: 1019 and 1107, respectively;(xxx) SEQ ID NOs: 1020 and 1108, respectively;(yyy) SEQ ID NOs: 1021 and 1109, respectively;(zzz) SEQ ID NOs: 1022 and 1110, respectively;(aaaa) SEQ ID NOs: 1023 and 1111, respectively;(bbbb) SEQ ID NOs: 1024 and 1112, respectively;(cccc) SEQ ID NOs: 1025 and 1113, respectively;(dddd) SEQ ID NOs: 1026 and 1114, respectively;(eeee) SEQ ID NOs: 1027 and 1115, respectively;(ffff) SEQ ID NOs: 1028 and 1116, respectively;(gggg) SEQ ID NOs: 1029 and 1117, respectively;(hhhh) SEQ ID NOs: 1030 and 1118, respectively;(iiii) SEQ ID NOs: 1031 and 1119, respectively; and,(jjjj) SEQ ID NOs: 1032 and 1120, respectively.

In some embodiments of the RNAi oligonucleotide:

(i) the sense strand comprises the sequence and all of the modificationsof5′-mCs-mU-mC-mA-mA-mG-mG-fA-fU-fU-fU-mC-mA-mG-mU-mU-mA-mU-mA-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 963), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-4O-mUs]-fUs-fAs-fU-fA-mA-fC-mU-mG-fA-mA-mA-mU-fC-mC-mU-mU-mG-mA-mGs-mGs-mG-3′(SEQ ID NO: 1051), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=Chem. formula 5

(ii) the sense strand comprises the sequence and all of themodifications of5′-mUs-mC-mA-mA-mG-mG-mA-fU-fU-fU-fC-mA-mG-mU-mU-mA-mU-mA-mA-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 964), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-4O-mUs]-fUs-fUs-fA-fU-mA-fA-mC-mU-fG-mA-mA-mA-fU-mC-mC-mU-mU-mG-mAs-mGs-mG-3′(SEQ ID NO: 1052), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=Chem. formula 5

(iii) the sense strand comprises the sequence and all of themodifications of5′-mAs-mG-mC-mA-mG-mC-mG-fU-fC-fC-fA-mC-mU-mC-mA-mG-mA-mG-mC-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 1006), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-4O-mUs]-fGs-fCs-fU-fC-mU-fG-mA-mG-fU-mG-mG-mA-fC-mG-mC-mU-mG-mC-mUs-mGs-mG-3′(SEQ ID NO: 1094), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=Chem. formula 5

(iv) the sense strand comprises the sequence and all of themodifications of5′-mAs-mU-mG-mU-mG-mC-mA-fC-fG-fA-fA-mU-mG-mA-mC-mU-mG-mU-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 1018), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-4O-mUs]-fAs-fAs-fC-fA-mG-fU-mC-mA-fU-mU-mC-mG-fU-mG-mC-mA-mC-mA-mUs-mGs-mG-3′(SEQ ID NO: 1106), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=Chem. formula 5

optionally wherein the oligonucleotide is a Dicer substrate.

In an aspect, the invention provides a pharmaceutical compositioncomprising the RNAi oligonucleotide according to the invention and apharmaceutically acceptable carrier, delivery agent or excipient.

In an aspect, the invention provides a kit comprising the RNAioligonucleotide according to the invention, an optional pharmaceuticallyacceptable carrier, and a package insert comprising instructions foradministration to a subject having a disease, disorder or conditionassociated with NR1H3 expression, optionally for the treatment ofnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), or systemic lupus erythematosus

In an aspect, the invention provides the use of the RNAi oligonucleotideaccording to the invention, or the pharmaceutical composition accordingto the invention, in the manufacture of a medicament for the treatmentof a disease, disorder or condition associated with NR1H3 expression,optionally for the treatment of non-alcoholic fatty liver disease(NAFLD), non-alcoholic steatohepatitis (NASH), or systemic lupuserythematosus, optionally for use in in combination with a secondcomposition or therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a drawing of an oligonucleotide comprising a nickedtetraloop structure.

FIG. 1B provides a graph depicting the percent (%) remaining human NR1H3mRNA in liver of mice exogenously expressing human NR1H3 (hydrodynamicinjection model) after treatment with GalNAc-conjugated NR1H3oligonucleotides. Three days post-dose mice were hydrodynamicallyinjected (HDI) with the human NR1H3 mRNA construct. 18 hours later,livers were collected and human and mouse NR1H3 mRNA levels weremeasured. Exons indicate the location on the mRNA where the constructtargets. Constructs tested in FIG. 1B were selected from the screendescribed in Example 2. White arrows indicate potential constructs fornon-human primate (NHP) studies.

FIG. 2 provides graphs depicting the percentage (%) of remaining humanor mouse NR1H3 mRNA in liver of mice exogenously expressing human NR1H3(hydrodynamic injection model) after treatment with GalNAc-conjugatedNR1H3 oligonucleotides designed based on sequences identified using thealgorithm described in Example 2. Mice were dosed subcutaneously with 2mg/kg of the indicated NR1H3-GalNAc construct. Three days post-dose micewere hydrodynamically injected (HDI) with the human NR1H3 mRNAconstruct. 18 hours later, livers were collected and human and mouseNR1H3 mRNA levels were measured. Exons indicate the location on the mRNAwhere the construct targets. Benchmark constructs (NR1H3-769 andNR1H3-1469) were selected from prior sequence screening conducted atDicerna.

FIG. 3 provides a graph depicting the percent (%) remaining human NR1H3mRNA in liver of mice exogenously expressing human NR1H3 (HDI model)after treatment with GalNAc-conjugated NR1H3 oligonucleotides.Constructs tested in FIG. 3 are a repeat assay validating the constructstested in FIG. 2 . White arrows indicate potential constructs fornon-human primate studies.

FIG. 4 provides a graph depicting the percent (%) remaining human NR1H3mRNA in liver of mice exogenously expressing human NR1H3 (HDI model)after treatment with GalNAc-conjugated NR1H3 oligonucleotides.Constructs tested in FIG. 1B and FIG. 2 were validated and repeated intwo assays (experiment 1 and experiment 2) using the same methods. Whitearrows indicate potential constructs for non-human primate studies.

FIG. 5 provides a schematic depicting the dosing scheme and specimencollection for treatment of non-human primates with GalNAc-conjugatedNR1H3 oligonucleotides. 1 mg/kg or 4 mg/kg are dosed once every 4 weeksfor four months (Q4W×4). Timing for liver biopsies and blood collectionis shown.

DETAILED DESCRIPTION

The liver X receptors α (LXRα; encoded by the NR1H3 gene) and β (LXRβ;encoded by NR1H2 gene) are nuclear receptors that function in theregulation of lipid and cholesterol homeostasis, as well as inflammation(Venkateswaran, A., et al., PROC. NATL. ACAD. SCI. U.S.A, (2000); 97:12097-102). LXRα is highly expressed in lipid metabolism-related organssuch as the liver, small intestine, kidney, spleen, adrenal gland, andadipose tissue, whereas LXRβ expression is distributed ubiquitously. Thehypothesis of specific inhibition of LXRα in the hepatocytes to treatNASH-related dyslipidemia is supported by several clinical andpre-clinical observations. Activation of LXRα increases plasma andhepatic TG and plasma LDLc, as was demonstrated in human subjectstreated with LXR agonist in a dose-dependent manner (Kirchgessner, T.G., et al., CELL METAB, (2016); 24(2): 223-33). Consistent with thisobservation, hepatic deletion of LXRα in mice lowers liver fat andreduces plasma triglycerides. (J. CLIN. INVEST. (2012); 122(5): 1688-99.

In addition, hepatic expression of LXRα is significantly upregulated inliver biopsies from NAFLD and NASH patients (Ahn, S. B., et al. DIG.DIS. SCI. (2014); 59: 2975-82). However, avoiding inhibition of LXRα inmacrophages is desirable as LXRα activation increases reversecholesterol transport (RCT) which prevents atherosclerosis (CURR. OPIN.INVESTIG. DRUGS. (2003); 4(9): 1053-8). Taken together, and withoutbeing bound by theory, antagonism/inhibition of LXRα specifically inhepatocytes (e.g., via NR1H3-targeted RNAi oligonucleotides) decreasesde novo lipogenesis with concomitant preservation of LXRα function inmacrophages and stellate cells for their positive roles in preventinginflammation and fibrosis respectively, thus representing a promisingapproach for treatment of NAFLD and/or NASH. (Higuchi, N., et al.,HEPATOL. RES. (2008); 38: 1122-29) (Repa, J. J.; and, Mangelsdorf, D. J.ANNU. REV. CELL DEV. BIOL. (2000); 16: 459-81). This approach may bebest managed by a specific and targeted reduction of the NR1H3expression in the liver while other organs, tissues or cells expressingNR1H3 are left essentially alone. In this sense the current inventionmay provide an improved modality of treatment given its specifictargeting of mRNA production in the liver.

According to some aspects, the disclosure provides oligonucleotides(e.g., RNAi oligonucleotides) that reduce NR1H3 expression in the liver.In some embodiments, the oligonucleotides provided herein are designedto treat diseases associated with NR1H3 expression in the liver, e.g.,non-alcoholic fatty liver disease (NAFLD), or non-alcoholicsteatohepatitis (NASH). In some respects, the disclosure providesmethods of treating a disease associated with NR1H3 expression byreducing NR1H3 expression in cells (e.g., cells of the liver) or inorgans (e.g., liver).

Oligonucleotide Inhibitors of NR1H3 Expression NR1H3 Target Sequences

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) is targeted to a target sequence comprising a NR1H3mRNA. In some embodiments, an oligonucleotide described herein istargeted to a target sequence within a NR1H3 mRNA sequence. In someembodiments, the oligonucleotide described herein corresponds to atarget sequence within a NR1H3 mRNA sequence. In some embodiments, theoligonucleotide, or a portion, fragment, or strand thereof (e.g., anantisense strand or a guide strand of a double-stranded (ds) RNAioligonucleotide) binds or anneals to a target sequence comprising NR1H3mRNA, thereby inhibiting NR1H3 expression.

In some embodiments, the oligonucleotide is targeted to a NR1H3 targetsequence for the purpose of inhibiting NR1H3 expression in vivo. In someembodiments, the amount or extent of inhibition of NR1H3 expression byan oligonucleotide targeted to a NR1H3 target sequence correlates withthe potency of the oligonucleotide. In some embodiments, the amount orextent of inhibition of NR1H3 expression by an oligonucleotide targetedto a NR1H3 target sequence correlates with the amount or extent oftherapeutic benefit in a subject or patient having a disease, disorderor condition associated with NR1H3 expression treated with theoligonucleotide.

Through examination of the nucleotide sequence of mRNAs encoding NR1H3,including mRNAs of multiple different species (e.g., human, cynomolgusmonkey, mouse, and rat; see, e.g., Example 2) and as a result of invitro and in vivo testing (see, e.g., Examples 2-5), it has beendiscovered that certain nucleotide sequences of NR1H3 mRNA are moreamenable than others to oligonucleotide-based inhibition and are thususeful as target sequences for the oligonucleotides herein. In someembodiments, a sense strand of an oligonucleotide (e.g., an RNAioligonucleotide) described herein comprises a NR1H3 target sequence. Insome embodiments, a portion or region of the sense strand of anoligonucleotide described herein (e.g., an RNAi oligonucleotide)comprises a NR1H3 target sequence. In some embodiments, a NR1H3 targetsequence comprises, or consists of, a sequence of any one of SEQ ID NOs:1-384. In some embodiments, a NR1H3 target sequence can consist of oneof the sequences set forth in SEQ ID NO: 92, 285, and/or 354.

NR1H3 Targeting Sequences

In some embodiments, the oligonucleotides herein (e.g., RNAioligonucleotides) have regions of complementarity to NR1H3 mRNA (e.g.,within a target sequence of NR1H3 mRNA) for purposes of targeting theNR1H3 mRNA in cells and inhibiting and/or reducing NR1H3 expression. Insome embodiments, the oligonucleotides herein comprise a NR1H3 targetingsequence (e.g., an antisense strand or a guide strand of a dsRNAioligonucleotide) having a region of complementarity that binds oranneals to a NR1H3 target sequence by complementary (Watson-Crick) basepairing. The targeting sequence or region of complementarity isgenerally of a suitable length and base content to enable binding orannealing of the oligonucleotide (or a strand thereof) to a NR1H3 mRNAfor purposes of inhibiting and/or reducing NR1H3 expression. In someembodiments, the targeting sequence or region of complementarity is atleast about 12, at least about 13, at least about 14, at least about 15,at least about 16, at least about 17, at least about 18, at least about19, at least about 20, at least about 21, at least about 22, at leastabout 23, at least about 24, at least about 25, at least about 26, atleast about 27, at least about 28, at least about 29, or at least about30 nucleotides in length. In some embodiments, the targeting sequence orregion of complementarity is about 12 to about 30 (e.g., 12 to 30, 12 to22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides inlength. In some embodiments, the targeting sequence or region ofcomplementarity is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In someembodiments, the targeting sequence or region of complementarity is 18nucleotides in length. In some embodiments, the targeting sequence orregion of complementarity is 19 nucleotides in length. In someembodiments, the targeting sequence or region of complementarity is 20nucleotides in length. In some embodiments, the targeting sequence orregion of complementarity is 21 nucleotides in length. In someembodiments, the targeting sequence or region of complementarity is 22nucleotides in length. In some embodiments, the targeting sequence orregion of complementarity is 23 nucleotides in length. In someembodiments, the targeting sequence or region of complementarity is 24nucleotides in length. In some embodiments, an oligonucleotide comprisesa target sequence or region of complementarity complementary to asequence of any one of SEQ ID NOs: 1-384. In some embodiments, anoligonucleotide comprises a target sequence or region of complementaritycomplementary to a sequence of any one of SEQ ID NOs: 1125-1511 and thetargeting sequence or region of complementarity is 18 nucleotides inlength. In some embodiments, an oligonucleotide comprises a targetsequence or region of complementarity complementary to a sequence of anyone of SEQ ID NOs: 1-384, and the targeting sequence or region ofcomplementarity is 19 nucleotides in length. In some embodiments, anoligonucleotide comprises a target sequence or region of complementaritycomplementary to a sequence of any one of SEQ ID NOs: 1125-1511, and thetargeting sequence or region of complementarity is 19 nucleotides inlength.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a targeting sequence or a region ofcomplementarity (e.g., an antisense strand or a guide strand of adouble-stranded oligonucleotide) that is fully complementary to a NR1H3target sequence. In some embodiments, the targeting sequence or regionof complementarity is partially complementary to a NR1H3 targetsequence. In some embodiments, the oligonucleotide comprises a targetingsequence or region of complementarity that is fully complementary to aNR1H3 target sequence. In some embodiments, the oligonucleotidecomprises a targeting sequence or region of complementarity that ispartially complementary to a NR1H3 target sequence.

In some embodiments, the oligonucleotide comprises a targeting sequenceor region of complementarity that is fully complementary to a sequenceof any one of SEQ ID NOs: 1-384. In some embodiments, theoligonucleotide comprises a targeting sequence or region ofcomplementarity that is fully complementary to a sequence of any one ofSEQ ID NOs: 1125-1511. In some embodiments, the oligonucleotidecomprises a targeting sequence or region of complementarity that isfully complementary to the sequence set forth in SEQ ID NO: 1409, 1509,1510 or 1511. In some embodiments, the oligonucleotide comprises atargeting sequence or region of complementarity that is partiallycomplementary to a sequence of any one of SEQ ID NOs: 1-384. In someembodiments, the oligonucleotide comprises a targeting sequence orregion of complementarity that is partially complementary to a sequenceof any one of SEQ ID NOs: 1125-1511. In some embodiments, theoligonucleotide comprises a targeting sequence or region ofcomplementarity that is partially complementary to the sequence setforth in SEQ ID NO: 1409, 1509, 1510 or 1511.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a targeting sequence or region ofcomplementarity that is complementary to a contiguous sequence ofnucleotides comprising a NR1H3 mRNA, wherein the contiguous sequence ofnucleotides is about 12 to about 30 nucleotides in length (e.g., 12 to30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In someembodiments, the oligonucleotide comprises a targeting sequence orregion of complementarity that is complementary to a contiguous sequenceof nucleotides comprising a NR1H3 mRNA, wherein the contiguous sequenceof nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleotides in length. In some embodiments, the oligonucleotidecomprises a targeting sequence or region of complementarity that iscomplementary to a contiguous sequence of nucleotides comprising a NR1H3mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotidesin length. In some embodiments, the oligonucleotide comprises atargeting sequence or region of complementarity that is complementary toa contiguous sequence of nucleotides comprising a NR1H3 mRNA, whereinthe contiguous sequence of nucleotides is 20 nucleotides in length.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384. In some embodiments, anoligonucleotide herein (e.g., an RNAi oligonucleotide) comprises atargeting sequence or a region of complementary that is complementary toa contiguous sequence of nucleotides of any one of SEQ ID NOs:1125-1511, optionally wherein the contiguous sequence of nucleotides is19 nucleotides in length. In some embodiments, the oligonucleotidecomprises a targeting sequence or a region of complementary that iscomplementary to a contiguous sequence of nucleotides of any one of SEQID NOs:1409, 1509, 1510 or 1511, wherein the contiguous sequence ofnucleotides is 19 nucleotides in length.

In some embodiments, a targeting sequence or region of complementarityof an oligonucleotide herein (e.g., an RNAi oligonucleotide) iscomplementary to a contiguous sequence of nucleotides of any one of SEQID NOs: 1-384. In some embodiments, a targeting sequence or region ofcomplementarity of an oligonucleotide herein (e.g., an RNAioligonucleotide) is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511. In some embodiments, atargeting sequence or region of complementarity of an oligonucleotideherein (e.g., an RNAi oligonucleotide) is complementary to a contiguoussequence of nucleotides of any one of SEQ ID NOs: 1-384 and spans theentire length of an antisense strand. In some embodiments, a targetingsequence or region of complementarity of the oligonucleotide iscomplementary to a contiguous sequence of nucleotides of SEQ ID NOs:1-384 and spans a portion of the entire length of an antisense strand.In some embodiments, a targeting sequence or region of complementarityof the oligonucleotide is complementary to a contiguous sequence ofnucleotides of SEQ ID NOs: 1125-1511 and spans a portion of the entirelength of an antisense strand. In some embodiments, a targeting sequenceor region of complementarity of the oligonucleotide is complementary toa contiguous sequence of nucleotides of SEQ ID NOs: 1125-1511 and spansa portion of the entire length of an antisense strand. In someembodiments, an oligonucleotide herein (e.g., an RNAi oligonucleotide)comprises a region of complementarity (e.g., on an antisense strand of adsRNA) that is at least partially (e.g., fully) complementary to acontiguous stretch of nucleotides spanning nucleotides 1-19 or 1-20 of asequence as set forth in any one of SEQ ID NOs: 1-384. In someembodiments, an oligonucleotide herein (e.g., an RNAi oligonucleotide)comprises a region of complementarity (e.g., on an antisense strand of adsRNA) that is at least partially (e.g., fully) complementary to acontiguous stretch of nucleotides spanning nucleotides 1-19 or 1-20 of asequence as set forth in any one of SEQ ID NOs: 1125-1511.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a targeting sequence or region ofcomplementarity having one or more base pair (bp) mismatches with thecorresponding NR1H3 target sequence. In some embodiments, the targetingsequence or region of complementarity may have up to about 1, up toabout 2, up to about 3, up to about 4, up to about 5, etc. mismatcheswith the corresponding NR1H3 target sequence provided that the abilityof the targeting sequence or region of complementarity to bind or annealto the NR1H3 mRNA under appropriate hybridization conditions and/or theability of the oligonucleotide to inhibit NR1H3 expression ismaintained. Alternatively, the targeting sequence or region ofcomplementarity may have no more than 1, no more than 2, no more than 3,no more than 4, or no more than 5 mismatches with the correspondingNR1H3 target sequence provided that the ability of the targetingsequence or region of complementarity to bind or anneal to the NR1H3mRNA under appropriate hybridization conditions and/or the ability ofthe oligonucleotide to inhibit NR1H3 expression is maintained. In someembodiments, the oligonucleotide comprises a targeting sequence orregion of complementarity having 1 mismatch with the correspondingtarget sequence. In some embodiments, the oligonucleotide comprises atargeting sequence or region of complementarity having 2 mismatches withthe corresponding target sequence. In some embodiments, theoligonucleotide comprises a targeting sequence or region ofcomplementarity having 3 mismatches with the corresponding targetsequence. In some embodiments, the oligonucleotide comprises a targetingsequence or region of complementarity having 4 mismatches with thecorresponding target sequence. In some embodiments, the oligonucleotidecomprises a targeting sequence or region of complementarity having 5mismatches with the corresponding target sequence. In some embodiments,the oligonucleotide comprises a targeting sequence or region ofcomplementarity having more than one mismatch (e.g., 2, 3, 4, 5 or moremismatches) with the corresponding target sequence, wherein at least 2(e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3,4, 5 or more mismatches in a row), or wherein the mismatches areinterspersed throughout the targeting sequence or region ofcomplementarity. In some embodiments, the oligonucleotide comprises atargeting sequence or region of complementarity having more than onemismatch (e.g., 2, 3, 4, 5 or more mismatches) with the correspondingtarget sequence, wherein at least 2 (e.g., all) of the mismatches arepositioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row),or wherein at least one or more non-mismatched base pair is locatedbetween the mismatches, or a combination thereof. In some embodiments,the oligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384, wherein the targetingsequence or region of complementarity may have up to about 1, up toabout 2, up to about 3, up to about 4, up to about 5, etc. mismatcheswith the corresponding NR1H3 target sequence. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511, wherein the targetingsequence or region of complementarity may have up to about 1, up toabout 2, up to about 3, up to about 4, up to about 5, etc. mismatcheswith the corresponding NR1H3 target sequence. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384, wherein the targetingsequence or region of complementarity may have no more than 1, no morethan 2, no more than 3, no more than 4, or no more than 5 mismatcheswith the corresponding NR1H3 target sequence. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511, wherein the targetingsequence or region of complementarity may have no more than 1, no morethan 2, no more than 3, no more than 4, or no more than 5 mismatcheswith the corresponding NR1H3 target sequence. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs:1409, 1509, 1510 or 1511, whereinthe targeting sequence or region of complementarity may have up to about1, up to about 2, up to about 3, up to about 4, up to about 5, etc.mismatches with the corresponding NR1H3 target sequence. In someembodiments, the oligonucleotide comprises a targeting sequence or aregion of complementary that is complementary to a contiguous sequenceof nucleotides of any one of SEQ ID NOs: 1409, 1509, 1510 or 1511,wherein the targeting sequence or region of complementarity may have nomore than 1, no more than 2, no more than 3, no more than 4, or no morethan 5 mismatches with the corresponding NR1H3 target sequence.

Types of Oligonucleotides

A variety of oligonucleotide types and/or structures are useful fortargeting NR1H3 in the methods herein including, but not limited to,RNAi oligonucleotides, antisense oligonucleotides (ASOs), miRNAs, etc.Any of the oligonucleotide types described herein or elsewhere arecontemplated for use as a framework to incorporate a NR1H3 targetingsequence herein for the purposes of inhibiting NR1H3 expression.

In some embodiments, the oligonucleotides herein inhibit NR1H3expression by engaging with RNA interference (RNAi) pathways upstream ordownstream of Dicer involvement. For example, RNAi oligonucleotides havebeen developed with each strand having sizes of about 19-25 nucleotideswith at least one 3′-overhang of 1 to 5 nucleotides (see, e.g., U.S.Pat. No. 8,372,968). Longer oligonucleotides also have been developedthat are processed by Dicer to generate active RNAi products (see, e.g.,U.S. Pat. No. 8,883,996). Further work produced extended dsRNAs where atleast one end of at least one strand is extended beyond a duplextargeting region, including structures where one of the strands includesa thermodynamically stabilizing tetraloop structure (see, e.g., U.S.Pat. Nos. 8,513,207 and 8,927,705, as well as Intl. Patent ApplicationPublication No. WO 2010/033225). Such structures may includesingle-stranded (ss) extensions (on one or both sides of the molecule)as well as double-stranded (ds) extensions.

In some embodiments, the oligonucleotides herein engage with the RNAipathway downstream of the involvement of Dicer (e.g., Dicer cleavage).In some embodiments, the oligonucleotides described herein are Dicersubstrates. In some embodiments, upon endogenous Dicer processing,double-stranded nucleic acids of 19-23 nucleotides in length capable ofreducing NR1H3 expression are produced. In some embodiments, theoligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides inlength) in the 3′ end of the sense strand. In some embodiments, theoligonucleotide (e.g., siRNA) comprises a 21-nucleotide guide strandthat is antisense to a target RNA and a complementary passenger strand,in which both strands anneal to form a 19-bp duplex and 2 nucleotideoverhangs at either or both 3′ ends. Longer oligonucleotide designs alsoare available including oligonucleotides having a guide strand of 23nucleotides and a passenger strand of 21 nucleotides, where there is ablunt end on the right side of the molecule (3′ end of passengerstrand/5′ end of guide strand) and a two nucleotide 3′-guide strandoverhang on the left side of the molecule (5′ end of the passengerstrand/3′ end of the guide strand). In such molecules, there is a 21 bpduplex region. See, e.g., U.S. Pat. Nos. 9,012,138; 9,012,621 and9,193,753.

In some embodiments, the oligonucleotides herein comprise sense andantisense strands that are both in the range of about 17 to 36 (e.g., 17to 36, 20 to 25, or 21-23) nucleotides in length. In some embodiments,the oligonucleotides described herein comprise an antisense strand of19-30 nucleotides in length and a sense strand of 19-50 nucleotides inlength, wherein the antisense and sense strands are separate strandswhich form an asymmetric duplex region having an overhang of 1-4nucleotides at the 3′ terminus of the antisense strand. In someembodiments, an oligonucleotide herein comprises a sense and antisensestrand that are both in the range of about 19-22 nucleotides in length.In some embodiments, the sense and antisense strands are of equallength. In some embodiments, an oligonucleotide comprises sense andantisense strands, such that there is a 3′-overhang on either the sensestrand or the antisense strand, or both the sense and antisense strand.In some embodiments, for oligonucleotides that have sense and antisensestrands that are both in the range of about 21-23 nucleotides in length,a 3′-overhang on the sense, antisense, or both sense and antisensestrands is 1 or 2 nucleotides in length. In some embodiments, theoligonucleotide has a guide strand of 22 nucleotides and a passengerstrand of 20 nucleotides, where there is a blunt end on the right sideof the molecule (3′ end of passenger strand/5′ end of guide strand) anda 2 nucleotide 3′-guide strand overhang on the left side of the molecule(5′ end of the passenger strand/3′ end of the guide strand). In suchmolecules, there is a 20 bp duplex region.

Other oligonucleotide designs for use with the compositions and methodsherein include: 16-mer siRNAs (see, e.g., NUCLEIC ACIDS IN CHEMISTRY ANDBIOLOGY. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs(e.g., having 19 bp or shorter stems; see, e.g., Moore et al. METHODSMOL. BIOL. (2010); 629: 141-158), blunt siRNAs (e.g., of 19 bps inlength; see, e.g., Kraynack & Baker RNA (2006); 12: 163-176),asymmetrical siRNAs (aiRNA; see, e.g., Sun et al. NAT. BIOTECHNOL.(2008); 26: 1379-1382), asymmetric shorter-duplex siRNA (see, e.g.,Chang et al. MOL. THER. (2009); 17: 725-32), fork siRNAs (see, e.g.,Hohjoh FEBS LETT. (2004); 557: 193-198), ss siRNAs (Elsner NAT.BIOTECHNOL. (2012); 30: 1063), dumbbell-shaped circular siRNAs (see,e.g., Abe et al. J. AM. CHEM. SOC. (2007); 129: 15108-09), and smallinternally segmented interfering RNA (siRNA; see, e.g., Bramsen et al.NUCLEIC ACIDS RES. (2007); 35: 5886-97). Further non-limiting examplesof an oligonucleotide structures that may be used in some embodiments toreduce or inhibit the expression of NR1H3 are microRNA (miRNA), shorthairpin RNA (shRNA) and short siRNA (see, e.g., Hamilton et al. EMBO J.(2002); 21: 4671-79; see also, US Patent Application Publication No.2009/0099115).

Still, in some embodiments, an oligonucleotide for reducing orinhibiting NR1H3 expression herein is single-stranded (ss). Suchstructures may include but are not limited to single-stranded RNAimolecules. Recent efforts have demonstrated the activity of ss RNAimolecules (see, e.g., Matsui et al. MOL. THER. (2016); 24: 946-55).However, in some embodiments, oligonucleotides herein are antisenseoligonucleotides (ASOs). An antisense oligonucleotide is asingle-stranded oligonucleotide that has a nucleobase sequence which,when written in the 5′ to 3′ direction, comprises the reverse complementof a targeted segment of a particular nucleic acid and is suitablymodified (e.g., as a gapmer) so as to induce RNaseH-mediated cleavage ofits target RNA in cells or (e.g., as a mixmer) so as to inhibittranslation of the target mRNA in cells. ASOs for use herein may bemodified in any suitable manner known in the art including, for example,as shown in U.S. Pat. No. 9,567,587 (including, e.g., length, sugarmoieties of the nucleobase (pyrimidine, purine), and alterations of theheterocyclic portion of the nucleobase). Further, ASOs have been usedfor decades to reduce expression of specific target genes (see, e.g.,Bennett et al. ANNU. REV. PHARMACOL. (2017); 57: 81-105).

In some embodiments, the antisense oligonucleotide shares a region ofcomplementarity with NR1H3 mRNA. In some embodiments, the antisenseoligonucleotide targets the human NR1H3 mRNA (Homo sapiens NR1H3, mRNA,transcript variant 5, NCBI Reference Sequence: NM_001251935.1). In someembodiments, the antisense oligonucleotide is 15-50 nucleotides inlength. In some embodiments, the antisense oligonucleotide is 15-25nucleotides in length. In some embodiments, the antisenseoligonucleotide is 22 nucleotides in length. In some embodiments, theantisense oligonucleotide is complementary to any one of SEQ ID NOs:1-384. In some embodiments, the antisense oligonucleotide iscomplementary to any one of SEQ ID NOs: 1125-1511. In some embodiments,the antisense oligonucleotide is at least 15 contiguous nucleotides inlength. In some embodiments, the antisense oligonucleotide is at least19 contiguous nucleotides in length. In some embodiments, the antisenseoligonucleotide is at least 20 contiguous nucleotides in length. In someembodiments, the antisense oligonucleotide differs by 1, 2, or 3nucleotides from the target sequence.

Double-Stranded Oligonucleotides

In some aspects, the disclosure provides double-stranded (ds) RNAioligonucleotides for targeting NR1H3 mRNA and inhibiting NR1H3expression (e.g., via the RNAi pathway) comprising a sense strand (alsoreferred to herein as a passenger strand) and an antisense strand (alsoreferred to herein as a guide strand). In some embodiments, the sensestrand and antisense strand are separate strands and are not covalentlylinked. In some embodiments, the sense strand and antisense strand arecovalently linked. In some embodiments, the sense strand and antisensestrand form a duplex region, wherein the sense strand and antisensestrand, or a portion thereof, binds with one another in a complementaryfashion (e.g., by Watson-Crick base pairing).

In some embodiments, the sense strand has a first region (R1) and asecond region (R2), wherein R2 comprises a first subregion (S1), atetraloop (L) or triloop (triL), and a second subregion (S2), wherein Lor triL is located between S1 and S2, and wherein S1 and S2 form asecond duplex (D2). D2 may have various length. In some embodiments, D2is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6,5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1,2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp inlength. In some embodiments, R1 of the sense strand and the antisensestrand form a first duplex (D1). In some embodiments, D1 is at leastabout 15 (e.g., at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, or at least 21) nucleotides in length. In someembodiments, D1 is in the range of about 12 to 30 nucleotides in length(e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to30, or 21 to 30 nucleotides in length). In some embodiments, D1 is atleast 12 nucleotides in length (e.g., at least 12, at least 15, at least20, at least 25, or at least 30 nucleotides in length). In someembodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, D1is 20 nucleotides in length. In some embodiments, D1 comprising sensestrand and antisense strand does not span the entire length of the sensestrand and/or antisense strand. In some embodiments, D1 comprising thesense strand and antisense strand spans the entire length of either thesense strand or antisense strand or both. In certain embodiments, D1comprising the sense strand and antisense strand spans the entire lengthof both the sense strand and the antisense strand.

In some embodiments, an oligonucleotide provided herein comprises asense strand having a sequence of any one of SEQ ID NOs: 1-384 and anantisense strand comprising a complementary sequence selected from SEQID NOs: 385-768. In some embodiments, an oligonucleotide provided hereincomprises a sense strand having a sequence of any one of SEQ ID NOs:1125-1511 and an antisense strand comprising a complementary sequenceselected from SEQ ID NOs: 1512-1515.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand having a sequence of any oneof SEQ ID NOs: 769-856 or 1519-1552 and an antisense strand comprising acomplementary sequence selected from SEQ ID NOs: 857-944 as is arrangedin Tables 3 and 4.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strandcomprising nucleotide sequences selected from:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strandcomprising nucleotide sequences selected from:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively.

In some embodiments, the sense strand comprises the sequence of SEQ IDNO: 786 and the antisense strand comprises the sequence of SEQ ID NO:874.

In some embodiments, the sense strand comprises the sequence of SEQ IDNO: 787 and the antisense strand comprises the sequence of SEQ ID NO:875.

In some embodiments, the sense strand comprises the sequence of SEQ IDNO: 1537 and the antisense strand comprises the sequence of SEQ ID NO:929.

In some embodiments, the sense strand comprises the sequence of SEQ IDNO: 813 and the antisense strand comprises the sequence of SEQ ID NO:901.

It should be appreciated that, in some embodiments, sequences presentedin the Sequence Listing may be referred to in describing the structureof an oligonucleotide (e.g., a dsRNAi oligonucleotide) or other nucleicacid. In such embodiments, the actual oligonucleotide or other nucleicacid may have one or more alternative nucleotides (e.g., an RNAcounterpart of a DNA nucleotide or a DNA counterpart of an RNAnucleotide) and/or one or more modified nucleotides and/or one or moremodified internucleotide linkages and/or one or more other modificationwhen compared with the specified sequence while retaining essentiallysame or similar complementary properties as the specified sequence.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a 25-nucleotide sense strand and a27-nucleotide antisense strand that when acted upon by a Dicer enzymeresults in an antisense strand that is incorporated into the matureRISC. In some embodiments, the 25-nucleotide sense strand comprises asequence selected from SEQ ID NOs: 1-384. In some embodiments, the27-nucleotide antisense strand comprises a sequence selected from SEQ IDNOs: 385-768. In some embodiments, the sense strand of theoligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 nucleotides). In some embodiments, the sense strand of theoligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or30 nucleotides). In some embodiments, the sense strand of theoligonucleotide comprises a nucleotide sequence selected from SEQ IDNOs: 769-856, wherein the nucleotide sequence is longer than 27nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides).). In someembodiments, the sense strand of the oligonucleotide comprises anucleotide sequence selected from SEQ ID NOs: 1519-1552, wherein thenucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 nucleotides). In some embodiments, the sense strand of theoligonucleotide comprises a nucleotide sequence selected from SEQ IDNOs: 769-856, wherein the nucleotide sequence is longer than 25nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

In some embodiments, oligonucleotides herein (e.g., RNAioligonucleotides) have one 5′ end that is thermodynamically less stablewhen compared to the other 5′ end. In some embodiments, an asymmetricoligonucleotide is provided that includes a blunt end at the 3′ end of asense strand and a 3′-overhang at the 3′ end of an antisense strand. Insome embodiments, the 3′-overhang on the antisense strand is about 1-8nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides inlength). In some embodiments, the oligonucleotide has an overhangcomprising two (2) nucleotides on the 3′ end of the antisense (guide)strand. However, other overhangs are possible. In some embodiments, anoverhang is a 3′-overhang comprising a length of between 1 and 6nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5,2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. However, in someembodiments, the overhang is a 5′-overhang comprising a length ofbetween 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2,2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5,5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. In someembodiments, the oligonucleotide comprises a targeting sequence or aregion of complementary that is complementary to a contiguous sequenceof nucleotides of any one of SEQ ID NOs: 1-384, and a 5′-overhangcomprising a length of between 1 and 6 nucleotides. In some embodiments,the oligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511, and a 5′-overhangcomprising a length of between 1 and 6 nucleotides. In some embodiments,the oligonucleotide comprises a sense strand comprising a nucleotidesequence selected from SEQ ID NOs: 769-856, wherein the oligonucleotidecomprises a 5′-overhang comprising a length of between 1 and 6nucleotides. In some embodiments, the oligonucleotide comprises a sensestrand comprising a nucleotide sequence selected from SEQ ID NOs:1519-1552, wherein the oligonucleotide comprises a 5′-overhangcomprising a length of between 1 and 6 nucleotides. In some embodiments,the oligonucleotide comprises an antisense strand comprising anucleotide sequence selected from SEQ ID NOs: 857-944, wherein theoligonucleotide comprises a 5′-overhang comprising a length of between 1and 6 nucleotides. In some embodiments, the oligonucleotide comprises asense strand comprising a nucleotide sequence selected from SEQ ID NOs:769-856 and antisense strand comprising a nucleotide sequence selectedfrom SEQ ID NOs: 857-944, wherein the oligonucleotide comprises a5′-overhang comprising a length of between 1 and 6 nucleotides. In someembodiments, the oligonucleotide comprises a sense strand comprising anucleotide sequence selected from SEQ ID NOs: 1519-1552 and antisensestrand comprising a nucleotide sequence selected from SEQ ID NOs:857-944, wherein the oligonucleotide comprises a 5′-overhang comprisinga length of between 1 and 6 nucleotides.

In some embodiments, two (2) terminal nucleotides on the 3′ end of anantisense strand are modified. In some embodiments, the two (2) terminalnucleotides on the 3′ end of the antisense strand are complementary withthe target mRNA (e.g., NR1H3 mRNA). In some embodiments, the two (2)terminal nucleotides on the 3′ end of the antisense strand are notcomplementary with the target mRNA. In some embodiments, the two (2)terminal nucleotides on the 3′ end of the antisense strand of anoligonucleotide herein are unpaired. In some embodiments, the two (2)terminal nucleotides on the 3′ end of the antisense strand of anoligonucleotide herein comprise an unpaired GG. In some embodiments, thetwo (2) terminal nucleotides on the 3′ end of an antisense strand of anoligonucleotide herein are not complementary to the target mRNA. In someembodiments, two (2) terminal nucleotides on each 3′ end of anoligonucleotide are GG. In some embodiments, one or both of the two (2)terminal GG nucleotides on each 3′ end of an oligonucleotide herein isnot complementary with the target mRNA. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384, wherein the two (2)terminal nucleotides on the 3′ end of the antisense strand of theoligonucleotide herein comprises an unpaired GG. In some embodiments,the oligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511, wherein the two (2)terminal nucleotides on the 3′ end of the antisense strand of theoligonucleotide herein comprises an unpaired GG. In some embodiments,the oligonucleotide comprises an antisense strand comprising anucleotide sequence selected from SEQ ID NOs: 857-944, wherein the two(2) terminal nucleotides on the 3′ end of the antisense strand of theoligonucleotide comprises an unpaired GG. In some embodiments, theoligonucleotide comprises a sense strand comprising a nucleotidesequence selected from SEQ ID NOs: 769-856 and antisense strandcomprising a nucleotide sequence selected from SEQ ID NOs: 857-944,wherein the two (2) terminal nucleotides on the 3′ end of the antisensestrand of the oligonucleotide comprises an unpaired GG. In someembodiments, the oligonucleotide comprises a sense strand comprising anucleotide sequence selected from SEQ ID NOs: 1519-1552 and antisensestrand comprising a nucleotide sequence selected from SEQ ID NOs:857-944, wherein the two (2) terminal nucleotides on the 3′ end of theantisense strand of the oligonucleotide comprises an unpaired GG.

In some embodiments, there is one or more (e.g., 1, 2, 3, 4, or 5)mismatch(s) between a sense and antisense strand comprising anoligonucleotide herein (e.g., an RNAi oligonucleotide). If there is morethan one mismatch between a sense and antisense strand, they may bepositioned consecutively (e.g., 2, 3 or more in a row), or interspersedthroughout the region of complementarity. In some embodiments, the 3′end of the sense strand comprises one or more mismatches. In someembodiments, two (2) mismatches are incorporated at the 3′ end of thesense strand. In some embodiments, base mismatches, or destabilizationof segments at the 3′ end of the sense strand of an oligonucleotideherein improves or increases the potency of the oligonucleotide. In someembodiments, the sense and antisense strands of an oligonucleotideherein comprise nucleotides sequences selected from the group consistingof:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein there is one or more (e.g., 1, 2, 3, 4 or 5) mismatch(s) betweenthe sense and antisense strands.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strandcomprising nucleotide sequences selected from:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,

wherein there is one or more (e.g., 1, 2, 3, 4, or 5) mismatch(s)between the sense and antisense strands.

Antisense Strands

In some embodiments, an antisense strand of an oligonucleotide herein(e.g., an RNAi oligonucleotide) is referred to as a “guide strand”. Forexample, an antisense strand that engages with RNA-induced silencingcomplex (RISC) and binds to an Argonaute protein such as Ago2, orengages with or binds to one or more similar factors, and directssilencing of a target gene, as the antisense strand is referred to as aguide strand. In some embodiments, a sense strand comprising a region ofcomplementary to a guide strand is referred to herein as a “passengerstrand.”

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises an antisense strand of up to about 50nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, upto 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotidesin length).

In some embodiments, an oligonucleotide comprises an antisense strand ofat least about 12 nucleotides in length (e.g., at least 12, at least 15,at least 19, at least 21, at least 22, at least 25, at least 27, atleast 30, at least 35, or at least 38 nucleotides in length). In someembodiments, an oligonucleotide comprises an antisense strand in a rangeof about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28,15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length.In some embodiments, an oligonucleotide comprises antisense strand of 15to 30 nucleotides in length. In some embodiments, an antisense strand ofany one of the oligonucleotides disclosed herein is of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In someembodiments, an oligonucleotide comprises an antisense strand of 22nucleotides in length.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) for targeting NR1H3 comprises an antisense strandcomprising or consisting of a sequence as set forth in any one of SEQ IDNOs: 385-768. In some embodiments, an oligonucleotide herein comprisesan antisense strand comprising at least about 12 (e.g., at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, or at least 23)contiguous nucleotides of a sequence as set forth in any one of SEQ IDNOs: 385-768. In some embodiments, an oligonucleotide disclosed hereinfor targeting NR1H3 comprises an antisense strand comprising orconsisting of a sequence as set forth in any one of SEQ ID NOs: 857-944.In some embodiments, an oligonucleotide herein comprises an antisensestrand comprising at least about 12 (e.g., at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, or at least 23) contiguousnucleotides of a sequence as set forth in any one of SEQ ID NOs:857-944. In some embodiments, an oligonucleotide disclosed herein fortargeting NR1H3 comprises an antisense strand comprising or consistingof a sequence as set forth in any one of SEQ ID NOs: 874, 875, 929, and901. In some embodiments, an oligonucleotide herein comprises anantisense strand comprising at least about 12 (e.g., at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22 or at least 23)contiguous nucleotides of a sequence as set forth in any one of SEQ IDNOs: 874, 875, 929, and 901.

In some embodiments, an oligonucleotide herein comprises an antisensestrand comprising a nucleotide sequence selected from SEQ ID NOs:1512-1515.

Sense Strands

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) for targeting NR1H3 mRNA and inhibiting NR1H3expression comprises a sense strand sequence as set forth in any one ofSEQ ID NOs: 1-384. In some embodiments, an oligonucleotide disclosedherein (e.g., an RNAi oligonucleotide) for targeting NR1H3 mRNA andinhibiting NR1H3 expression comprises a sense strand sequence as setforth in any one of SEQ ID NOs: 1125-1511. In some embodiments, anoligonucleotide herein has a sense strand comprised of at least about 12(e.g., at least 13, at least 14, at least 15, at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, or atleast 23) contiguous nucleotides of a sequence as set forth in in anyone of SEQ ID NOs: 1-384. In some embodiments, an oligonucleotide hereinhas a sense strand comprised of at least about 12 (e.g., at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22 or at least 23) contiguousnucleotides of a sequence as set forth in in any one of SEQ ID NOs:1125-1511. In some embodiments, an oligonucleotide disclosed herein fortargeting NR1H3 mRNA and inhibiting NR1H3 expression comprises a sensestrand sequence as set forth in any one of SEQ ID NOs: 769-856. In someembodiments, an oligonucleotide disclosed herein for targeting NR1H3mRNA and inhibiting NR1H3 expression comprises a sense strand sequenceas set forth in any one of SEQ ID NOs: 1519-1552. In some embodiments,an oligonucleotide herein has a sense strand comprised of least about 12(e.g., at least 13, at least 14, at least 15, at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, or atleast 23) contiguous nucleotides of a sequence as set forth in any oneof SEQ ID NOs: 769-856. In some embodiments, an oligonucleotide hereinhas a sense strand comprised of least about 12 (e.g., at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, or at least 23) contiguousnucleotides of a sequence as set forth in any one of SEQ ID NOs:1519-1552. In some embodiments, an oligonucleotide disclosed herein fortargeting NR1H3 mRNA and inhibiting NR1H3 expression comprises a sensestrand sequence as set forth in any one of SEQ ID NOs: 786, 787, 1537,and 813. In some embodiments, an oligonucleotide herein has a sensestrand that comprise at least about 12 (e.g., at least 13, at least 14,at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, or at least 23) contiguousnucleotides of a sequence as set forth in any one of SEQ ID NOs: 786,787, 1537, and 813. In some embodiments, an oligonucleotide disclosedherein for targeting NR1H3 mRNA and inhibiting NR1H3 expressioncomprises a sense strand sequence as set forth in any one of SEQ ID NOs:1409, 1509, 1510 and 1511. In some embodiments, an oligonucleotideherein has a sense strand that comprise at least about 12 (e.g., atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22 or at least 23)contiguous nucleotides of a sequence as set forth in any one of SEQ IDNOs: 1409, 1509, 1510 and 1511.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand (or passenger strand) of up toabout 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, upto 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12nucleotides in length). In some embodiments, an oligonucleotide hereincomprises a sense strand of at least about 12 nucleotides in length(e.g., at least 12, at least 15, at least 19, at least 21, at least 25,at least 27, at least 30, at least 36, or at least 38 nucleotides inlength). In some embodiments, an oligonucleotide herein comprises asense strand in a range of about 12 to about 50 (e.g., 12 to 50, 12 to40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to40, or 32 to 40) nucleotides in length. In some embodiments, anoligonucleotide herein comprises a sense strand of 15 to 50 nucleotidesin length. In some embodiments, an oligonucleotide herein comprises asense strand of 18 to 36 nucleotides in length. In some embodiments, anoligonucleotide herein comprises a sense strand of 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length. In some embodiments, an oligonucleotide hereincomprises a sense strand of 36 nucleotides in length.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand comprising a stem-loopstructure at the 3′ end of the sense strand. In some embodiments, thestem-loop is formed by intrastrand base pairing. In some embodiments, asense strand comprises a stem-loop structure at its 5′ end. In someembodiments, the stem of the stem-loop comprises a duplex of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In someembodiments, the stem of the stem-loop comprises a duplex of 2nucleotides in length. In some embodiments, the stem of the stem-loopcomprises a duplex of 3 nucleotides in length. In some embodiments, thestem of the stem-loop comprises a duplex of 4 nucleotides in length. Insome embodiments, the stem of the stem-loop comprises a duplex of 5nucleotides in length. In some embodiments, the stem of the stem-loopcomprises a duplex of 6 nucleotides in length. In some embodiments, thestem of the stem-loop comprises a duplex of 7 nucleotides in length. Insome embodiments, the stem of the stem-loop comprises a duplex of 8nucleotides in length. In some embodiments, the stem of the stem-loopcomprises a duplex of 9 nucleotides in length. In some embodiments, thestem of the stem-loop comprises a duplex of 10 nucleotides in length. Insome embodiments, the stem of the stem-loop comprises a duplex of 11nucleotides in length. In some embodiments, the stem of the stem-loopcomprises a duplex of 12 nucleotides in length. In some embodiments, thestem of the stem-loop comprises a duplex of 13 nucleotides in length. Insome embodiments, the stem of the stem-loop comprises a duplex of 14nucleotides in length.

In some embodiments, a stem-loop provides the oligonucleotide protectionagainst degradation (e.g., enzymatic degradation), facilitates orimproves targeting and/or delivery to a target cell, tissue, or organ(e.g., the liver), or both. For example, in some embodiments, the loopof a stem-loop is comprised of nucleotides comprising one or moremodifications that facilitate, improve, or increase targeting to atarget mRNA (e.g., a NR1H3 mRNA), inhibition of target gene expression(e.g., NR1H3 expression), and/or delivery, uptake, and/or penetranceinto a target cell, tissue, or organ (e.g., the liver), or a combinationthereof. In some embodiments, the stem-loop itself or modification(s) tothe stem-loop do not affect or do not substantially affect the inherentgene expression inhibition activity of the oligonucleotide, butfacilitates, improves, or increases stability (e.g., provides protectionagainst degradation) and/or delivery, uptake, and/or penetrance of theoligonucleotide to a target cell, tissue, or organ (e.g., the liver). Incertain embodiments, an oligonucleotide herein comprises a sense strandcomprising (e.g., at its 3′ end) a stem-loop set forth as: S1-L-S2, inwhich S1 is complementary to S2, and in which L forms a single-strandedloop of linked nucleotides between S1 and S2 of up to about 10nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides inlength). In some embodiments, the loop (L) is 3 nucleotides in length.In some embodiments, the loop (L) is 4 nucleotides in length. In someembodiments, the loop (L) is 5 nucleotides in length. In someembodiments, the loop (L) is 6 nucleotides in length. In someembodiments, the loop (L) is 7 nucleotides in length. In someembodiments, the loop (L) is 8 nucleotides in length. In someembodiments, the loop (L) is 9 nucleotides in length. In someembodiments, the loop (L) is 10 nucleotides in length.

In some embodiments, the tetraloop comprises the sequence 5′-GAAA-3′. Insome embodiments, the stem loop comprises the sequence5′-GCAGCCGAAAGGCUGC-3′ (SEQ ID NO: 1121).

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384, and the oligonucleotidecomprises a sense strand comprising (e.g., at its 3′ end) a stem-loopset forth as: S1-L-S2, in which S1 is complementary to S2, and in whichL forms a single-stranded loop between S1 and S2 of up to about 10nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength). In some embodiments, an oligonucleotide provided herein (e.g.,an RNAi oligonucleotide) comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1125-1511, and the oligonucleotidecomprises a sense strand comprising (e.g., at its 3′ end) a stem-loopset forth as: S1-L-S2, in which S1 is complementary to S2, and in whichL forms a single-stranded loop between S1 and S2 of up to about 10nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength). In some embodiments, the oligonucleotide comprises a targetingsequence or a region of complementary that is complementary to acontiguous sequence of nucleotides of any one of SEQ ID NOs: 1-384, andthe oligonucleotide comprises a sense strand comprising (e.g., at its 3′end) a stem-loop set forth as: S1-L-S2, in which S1 is complementary toS2, and in which L forms a single-stranded loop between S1 and S2 of 4nucleotides in length.

In some embodiments, the oligonucleotide comprises a targeting sequenceor a region of complementary that is complementary to a contiguoussequence of nucleotides of any one of SEQ ID NOs: 1-384, and theoligonucleotide comprises a sense strand comprising (e.g., at its 3′end) a stem-loop set forth as: S1-L-S2, in which S1 is complementary toS2, and in which L forms a single-stranded loop between S1 and S2 of 4nucleotides in length. In some embodiments, the oligonucleotidecomprises a targeting sequence or a region of complementary that iscomplementary to a contiguous sequence of nucleotides of any one of SEQID NOs: 1125-1511, and the oligonucleotide comprises a sense strandcomprising (e.g., at its 3′ end) a stem-loop set forth as: S1-L-S2, inwhich S1 is complementary to S2, and in which L forms a single-strandedloop between S1 and S2 of 4 nucleotides in length.

In some embodiments, a loop (L) of a stem-loop having the structureS1-L-S2 as described herein is a triloop. In some embodiments, theoligonucleotide comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384 and a triloop. In someembodiments, the triloop comprises ribonucleotides,deoxyribonucleotides, modified nucleotides, ligands (e.g., deliveryligands), and combinations thereof. In some embodiments, a loop (L) of astem-loop having the structure S1-L-S2 as described herein is a triloop.In some embodiments, the oligonucleotide comprises a targeting sequenceor a region of complementary that is complementary to a contiguoussequence of nucleotides of any one of SEQ ID NOs: 1125-1511 and atriloop. In some embodiments, the triloop comprises ribonucleotides,deoxyribonucleotides, modified nucleotides, ligands (e.g., deliveryligands), and combinations thereof.

In some embodiments, a loop (L) of a stem-loop having the structureS1-L-S2 as described above is a tetraloop. In some embodiments, anoligonucleotide herein comprises a targeting sequence or a region ofcomplementary that is complementary to a contiguous sequence ofnucleotides of any one of SEQ ID NOs: 1-384 and a tetraloop. In someembodiments, the tetraloop comprises ribonucleotides,deoxyribonucleotides, modified nucleotides, ligands (e.g., deliveryligands), and combinations thereof. In some embodiments, a loop (L) of astem-loop having the structure S1-L-S2 as described above is atetraloop. In some embodiments, an oligonucleotide herein comprises atargeting sequence or a region of complementary that is complementary toa contiguous sequence of nucleotides of any one of SEQ ID NOs: 1125-1511and a tetraloop. In some embodiments, the tetraloop comprisesribonucleotides, deoxyribonucleotides, modified nucleotides, ligands(e.g., delivery ligands), and combinations thereof.

Duplex Length

In some embodiments, a duplex formed between a sense and antisensestrand is at least 12 (e.g., at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, or at least 21) nucleotides inlength. In some embodiments, a duplex formed between a sense andantisense strand is in the range of 12-30 nucleotides in length (e.g.,12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In someembodiments, a duplex formed between a sense and antisense strand is 12,13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 12 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 13nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 14 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 15nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 16 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 17nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 18 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 19nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 20 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 21nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 22 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 23nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 24 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 25nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 26 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 27nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 28 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand is 29nucleotides in length. In some embodiments, a duplex formed between asense and antisense strand is 30 nucleotides in length. In someembodiments, a duplex formed between a sense and antisense strand doesnot span the entire length of the sense strand and/or antisense strand.In some embodiments, a duplex between a sense and antisense strand spansthe entire length of either the sense or antisense strands. In someembodiments, a duplex between a sense and antisense strand spans theentire length of both the sense strand and the antisense strand. In someembodiments, the sense and antisense strands of an oligonucleotidecomprise nucleotides sequences selected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein a duplex formed between a sense and antisense strand is in therange of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to30, or 21 to 30 nucleotides in length)

In some embodiments, a duplex between a sense and antisense strand spansthe entire length of both the sense strand and the antisense strand. Insome embodiments, the sense and antisense strands of an oligonucleotidecomprise nucleotides sequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,wherein a duplex formed between a sense and antisense strand is in therange of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to30, or 21 to 30 nucleotides in length)

Oligonucleotide Termini

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the termini of either or both strands comprise a blunt end. Insome embodiments, an oligonucleotide herein comprises sense andantisense strands that are separate strands which form an asymmetricduplex region having an overhang at the 3′ terminus of the antisensestrand. In some embodiments, an oligonucleotide herein comprises a sensestrand and an antisense strand, wherein the termini of either or bothstrands comprise an overhang comprising one or more nucleotides. In someembodiments, the one or more nucleotides comprising the overhang areunpaired nucleotides. In some embodiments, an oligonucleotide hereincomprises a sense strand and an antisense strand, wherein the 3′ terminiof the sense strand and the 5′ termini of the antisense strand comprisea blunt end. In some embodiments, an oligonucleotide herein comprises asense strand and an antisense strand, wherein the 5′ termini of thesense strand and the 3′ termini of the antisense strand comprise a bluntend.

In some embodiments, an oligonucleotide herein comprises a sense strandand an antisense strand, wherein the 3′ terminus of either or bothstrands comprise a 3′-overhang comprising one or more nucleotides. Insome embodiments, an oligonucleotide herein comprises a sense strand andan antisense strand, wherein the sense strand comprises a 3′-overhangcomprising one or more nucleotides. In some embodiments, anoligonucleotide herein comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 3′-overhang comprising one ormore nucleotides. In some embodiments, an oligonucleotide hereincomprises a sense strand and an antisense strand, wherein both the sensestrand and the antisense strand comprises a 3′-overhang comprising oneor more nucleotides.

In some embodiments, the 3′-overhang is about one (1) to twenty (20)nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). Insome embodiments, the 3′-overhang is about one (1) to nineteen (19), one(1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen(16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) tothirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1)to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven(7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one(1) to three (3), or about one (1) to two (2) nucleotides in length. Insome embodiments, the 3′-overhang is (1) nucleotide in length. In someembodiments, the 3′-overhang is two (2) nucleotides in length. In someembodiments, the 3′-overhang is three (3) nucleotides in length. In someembodiments, the 3′-overhang is four (4) nucleotides in length. In someembodiments, the 3′-overhang is five (5) nucleotides in length. In someembodiments, the 3′-overhang is six (6) nucleotides in length. In someembodiments, the 3′-overhang is seven (7) nucleotides in length. In someembodiments, the 3′-overhang is eight (8) nucleotides in length. In someembodiments, the 3′-overhang is nine (9) nucleotides in length. In someembodiments, the 3′-overhang is ten (10) nucleotides in length. In someembodiments, the 3′-overhang is eleven (11) nucleotides in length. Insome embodiments, the 3′-overhang is twelve (12) nucleotides in length.In some embodiments, the 3′-overhang is thirteen (13) nucleotides inlength. In some embodiments, the 3′-overhang is fourteen (14)nucleotides in length. In some embodiments, the 3′-overhang is fifteen(15) nucleotides in length. In some embodiments, the 3′-overhang issixteen (16) nucleotides in length. In some embodiments, the 3′-overhangis seventeen (17) nucleotides in length. In some embodiments, the3′-overhang is eighteen (18) nucleotides in length. In some embodiments,the 3′-overhang is nineteen (19) nucleotides in length. In someembodiments, the 3′-overhang is twenty (20) nucleotides in length.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 3′-overhang, wherein the senseand antisense strands of the oligonucleotide comprise nucleotidessequences selected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,and wherein the antisense strand comprises a 3′-overhang about one (1)to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides inlength), optionally wherein the 3′-overhang is two (2) nucleotides inlength.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 3′-overhang, wherein the senseand antisense strands of the oligonucleotide comprise nucleotidessequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,and wherein the antisense strand comprises a 3′-overhang about one (1)to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides inlength), optionally wherein the 3′-overhang is two (2) nucleotides inlength.

In some embodiments, an oligonucleotide herein comprises a sense strandand an antisense strand, wherein the 5′ terminus of either or bothstrands comprise a 5′-overhang comprising one or more nucleotides. Insome embodiments, an oligonucleotide herein comprises a sense strand andan antisense strand, wherein the sense strand comprises a 5′-overhangcomprising one or more nucleotides. In some embodiments, anoligonucleotide herein comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 5′-overhang comprising one ormore nucleotides. In some embodiments, an oligonucleotide hereincomprises a sense strand and an antisense strand, wherein both the sensestrand and the antisense strand comprises a 5′-overhang comprising oneor more nucleotides.

In some embodiments, the 5′-overhang is about one (1) to twenty (20)nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). Insome embodiments, the 5′-overhang is about one (1) to nineteen (19), one(1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen(16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) tothirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1)to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven(7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one(1) to three (3), or about one (1) to two (2) nucleotides in length. Insome embodiments, the 5′-overhang is (1) nucleotide in length. In someembodiments, the 5′-overhang is two (2) nucleotides in length. In someembodiments, the 5′-overhang is three (3) nucleotides in length. In someembodiments, the 5′-overhang is four (4) nucleotides in length. In someembodiments, the 5′-overhang is five (5) nucleotides in length. In someembodiments, the 5′-overhang is six (6) nucleotides in length. In someembodiments, the 5′-overhang is seven (7) nucleotides in length. In someembodiments, the 5′-overhang is eight (8) nucleotides in length. In someembodiments, the 5′-overhang is nine (9) nucleotides in length. In someembodiments, the 5′-overhang is ten (10) nucleotides in length. In someembodiments, the 5′-overhang is eleven (11) nucleotides in length. Insome embodiments, the 5′-overhang is twelve (12) nucleotides in length.In some embodiments, the 5′-overhang is thirteen (13) nucleotides inlength. In some embodiments, the 5′-overhang is fourteen (14)nucleotides in length. In some embodiments, the 5′-overhang is fifteen(15) nucleotides in length. In some embodiments, the 5′-overhang issixteen (16) nucleotides in length. In some embodiments, the 5′-overhangis seventeen (17) nucleotides in length. In some embodiments, the5′-overhang is eighteen (18) nucleotides in length. In some embodiments,the 5′-overhang is nineteen (19) nucleotides in length. In someembodiments, the 5′-overhang is twenty (20) nucleotides in length.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 5′-overhang, wherein the senseand antisense strands of the oligonucleotide comprise nucleotidessequences selected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,and wherein the antisense strand comprises a 3′-overhang about one (1)to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides inlength), optionally wherein the 3′-overhang is two (2) nucleotides inlength.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 5′-overhang, wherein the senseand antisense strands of the oligonucleotide comprise nucleotidessequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,and wherein the antisense strand comprises a 5′-overhang about one (1)to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides inlength), optionally wherein the 5′-overhang is two (2) nucleotides inlength.

In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) nucleotidescomprising the 3′ terminus or 5′ terminus of a sense and/or antisensestrand are modified. For example, in some embodiments, one or twoterminal nucleotides of the 3′ terminus of the antisense strand aremodified. In some embodiments, the last nucleotide at the 3′ terminus ofan antisense strand is modified, e.g., comprises 2′ modification, e.g.,a 2′-O-methoxyethyl. In some embodiments, an oligonucleotide providedherein comprises a sense strand having the sugar moiety at positions1-7, 12-27 and 31-36 modified with 2′OMe. In some embodiments, the lastone or two terminal nucleotides at the 3′ terminus of an antisensestrand are complementary with the target. In some embodiments, the lastone or two nucleotides at the 3′ terminus of the antisense strand arenot complementary with the target.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the 3′ terminus of the sense strand comprises a stem-loopdescribed herein (see FIG. 1A) and the 3′ terminus of the antisensestrand comprises a 3′-overhang described herein. In some embodiments, anoligonucleotide herein (e.g., an RNAi oligonucleotide) comprises a sensestrand and an antisense strand that form a nicked tetraloop structuredescribed herein (see FIG. 1A), wherein the 3′ terminus of the sensestrand comprises a stem-loop, wherein the loop is a tetraloop describedherein, and wherein the 3′ terminus of the antisense strand comprises a3′-overhang described herein (see FIG. 1A). In some embodiments, the3′-overhang is two (2) nucleotides in length. In some embodiments, thetwo (2) nucleotides comprising the 3′-overhang both comprise guanine (G)nucleobases. Typically, one or both of the nucleotides comprising the3′-overhang of the antisense strand are not complementary with thetarget mRNA.

Oligonucleotide Modifications

In some embodiments, an oligonucleotide described herein (e.g., an RNAioligonucleotide) comprises a modification. Oligonucleotides (e.g., RNAioligonucleotides) may be modified in various ways to improve or controlspecificity, stability, delivery, bioavailability, resistance fromnuclease degradation, immunogenicity, base-pairing properties, RNAdistribution and cellular uptake and other features relevant totherapeutic or research use.

In some embodiments, the modification is a modified sugar. In someembodiments, the modification is a 5′-terminal phosphate group. In someembodiments, the modification is a modified internucleotide linkage. Insome embodiments, the modification is a modified base. In someembodiments, an oligonucleotide described herein can comprise any one ofthe modifications described herein or any combination thereof. Forexample, in some embodiments, an oligonucleotide described hereincomprises at least one modified sugar, a 5′-terminal phosphate group, atleast one modified internucleotide linkage, and at least one modifiedbase. In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises at least one modified sugar, a5′-terminal phosphate group, at least one modified internucleotidelinkage, and at least one modified base.

In some embodiments, an oligonucleotide described herein comprises atleast one modified sugar, a 5′-terminal phosphate group, at least onemodified internucleotide linkage, and at least one modified base. Insome embodiments, the sense and antisense strands of an oligonucleotidecomprise nucleotides sequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises at least one modified sugar, a5′-terminal phosphate group, at least one modified internucleotidelinkage, and at least one modified base.

The number of modifications on an oligonucleotide (e.g., an RNAioligonucleotide) and the position of those nucleotide modifications mayinfluence the properties of an oligonucleotide. For example,oligonucleotides may be delivered in vivo by conjugating them to orencompassing them in a lipid nanoparticle (LNP) or similar carrier.However, when an oligonucleotide is not protected by an LNP or similarcarrier, it may be advantageous for at least some of the nucleotides tobe modified. Accordingly, in some embodiments, all or substantially allthe nucleotides of an oligonucleotide are modified. In some embodiments,more than half of the nucleotides are modified. In some embodiments,less than half of the nucleotides are modified. In some embodiments, thesugar moiety of all nucleotides comprising the oligonucleotide ismodified at the 2′ position. The modifications may be reversible orirreversible. In some embodiments, an oligonucleotide as disclosedherein has a number and type of modified nucleotides sufficient to causethe desired characteristics (e.g., protection from enzymaticdegradation, capacity to target a desired cell after in vivoadministration, and/or thermodynamic stability).

Sugar Modifications

In some embodiments, an oligonucleotide described herein (e.g., an RNAioligonucleotide) comprises a modified sugar. In some embodiments, amodified sugar (also referred herein to a sugar analog) includes amodified deoxyribose or ribose moiety in which, for example, one or moremodifications occur at the 2′, 3′, 4′ and/or 5′ carbon position of thesugar. In some embodiments, a modified sugar may also includenon-natural alternative carbon structures such as those present inlocked nucleic acids (“LNA”; see, e.g., Koshkin et al. TETRAHEDON(1998); 54: 3607-30), unlocked nucleic acids (“UNA”; see, e.g., Snead etal. MOL. THER-NUCL. ACIDS (2013); 2: e103) and bridged nucleic acids(“BNA”; see, e.g., Imanishi & Obika CHEM. COMMUN. (CAMB) (2002); 21:1653-59).

In some embodiments, a nucleotide modification in a sugar comprises a2′-modification. In some embodiments, a 2′-modification may be2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-fluoro (2′-F),2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA) or2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). In someembodiments, the modification is 2′-F, 2′-OMe or 2′-MOE. In someembodiments, a modification in a sugar comprises a modification of thesugar ring, which may comprise modification of one or more carbons ofthe sugar ring. For example, a modification of a sugar of a nucleotidemay comprise a 2′-oxygen of a sugar is linked to a 1′-carbon or4′-carbon of the sugar, or a 2′-oxygen is linked to the 1′-carbon or4′-carbon via an ethylene or methylene bridge. In some embodiments, amodified nucleotide has an acyclic sugar that lacks a 2′-carbon to3′-carbon bond. In some embodiments, a modified nucleotide has a thiolgroup, e.g., in the 4′ position of the sugar.

In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide)described herein comprises at least about 1 modified nucleotide (e.g.,at least 1, at least 5, at least 10, at least 15, at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 55, at least 60, or more). In some embodiments, the sense strandof the oligonucleotide comprises at least about 1 modified nucleotide(e.g., at least 1, at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, or more). In some embodiments, theantisense strand of the oligonucleotide comprises at least about 1modified nucleotide (e.g., at least 1, at least 5, at least 10, at least15, at least 20, or more).

In some embodiments, all the nucleotides of the sense strand of theoligonucleotide are modified. In some embodiments, all the nucleotidesof the antisense strand of the oligonucleotide are modified. In someembodiments, all the nucleotides of the oligonucleotide (i.e., both thesense strand and the antisense strand) are modified. In someembodiments, the modified nucleotide comprises a 2′-modification (e.g.,a 2′-F or 2′-OMe, 2′-MOE, and 2′-deoxy-2′-fluoro-β-d-arabinonucleicacid).

In some embodiments, the disclosure provides oligonucleotides havingdifferent modification patterns. In some embodiments, an oligonucleotideherein comprises a sense strand having a modification pattern as setforth in the Examples and Sequence Listing and an antisense strandhaving a modification pattern as set forth in the Examples and SequenceListing.

In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAioligonucleotide) comprises an antisense strand having nucleotides thatare modified with 2′-F. In some embodiments, an oligonucleotide hereincomprises an antisense strand comprising nucleotides that are modifiedwith 2′-F and 2′-OMe. In some embodiments, an oligonucleotide disclosedherein comprises a sense strand having nucleotides that are modifiedwith 2′-F. In some embodiments, an oligonucleotide disclosed hereincomprises a sense strand comprises nucleotides that are modified with2′-F and 2′-OMe.

In some embodiments, an oligonucleotide described herein comprises asense strand with about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of thenucleotides of the sense strand comprising a 2′-fluoro modification. Insome embodiments, about 11% of the nucleotides of the sense strandcomprise a 2-fluoro modification. In some embodiments, anoligonucleotide described herein comprises an antisense strand withabout 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%of the nucleotides of the antisense strand comprising a 2′-fluoromodification. In some embodiments, about 32% of the nucleotides of theantisense strand comprise a 2′-fluoro modification. In some embodiments,the oligonucleotide has about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, or 25% of its nucleotides comprising a 2′-fluoromodification. In some embodiments, about 19% of the nucleotides in thedsRNAi oligonucleotide comprise a 2′-fluoro modification.

In some embodiments, one or more of positions 8, 9, 10, or 11 of thesense strand is modified with a 2′-F group. In some embodiments, one ormore of positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand ismodified with a 2′-F group. In some embodiments, one or more ofpositions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modifiedwith a 2′-F group. In some embodiments, one or more of positions 2, 3,4, 5, 7, 8, 10, 14, 16, and 19 is modified with a 2′-F group. In someembodiments, the sugar moiety at each of nucleotides at positions 1-7and 12-20 in the sense strand is modified with a 2′-OMe. In someembodiments, the sugar moiety at each of nucleotides at positions 1-7,12-27, and 31-36 in the sense strand is modified with a 2′-OMe. In someembodiments, the sugar moiety at each of nucleotides at positions 6, 9,11-13, 15, 17, 18, and 20-22 in the sense strand is modified with a2′-OMe.

In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein one or more of positions 8, 9, 10 or 11 of the sense strand ismodified with a 2′-F group.

In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively, and;(d) SEQ ID NOs: 813 and 901, respectively,wherein one or more of positions 8, 9, 10, or 11 of the sense strand ismodified with a 2′-F group.

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 5, and 14 of the antisense strand modified with 2′-F andthe sugar moiety of each of the remaining nucleotides of the antisensestrand modified with a modification selected from the group consistingof 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl(EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 1, 2, 5, and 14 of the antisense strand modified with 2′-F andthe sugar moiety of each of the remaining nucleotides of the antisensestrand modified with a modification selected from the group consistingof 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl(EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 4, 5, and 14 of the antisense strand modified with 2′-F andthe sugar moiety of each of the remaining nucleotides of the antisensestrand modified with a modification selected from the group consistingof 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl(EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 1, 2, 3, 5, 7, and 14 of the antisense strand modified with2′-F and the sugar moiety of each of the remaining nucleotides of theantisense strand modified with a modification selected from the groupconsisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 3, 4, 5, 7, and 14 of the antisense strand modified with2′-F and the sugar moiety of each of the remaining nucleotides of theantisense strand modified with a modification selected from the groupconsisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 1, 2, 3, 5, 10, and 14 of the antisense strand modified with2′-F and the sugar moiety of each of the remaining nucleotides of theantisense strand modified with a modification selected from the groupconsisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 3, 4, 5, 10, and 14 of the antisense strand modified with2′-F and the sugar moiety of each of the remaining nucleotides of theantisense strand modified with a modification selected from the groupconsisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 3, 5, 7, 10, and 14 of the antisense strand modified with2′-F and the sugar moiety of each of the remaining nucleotides of theantisense strand modified with a modification selected from the groupconsisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modifiedwith 2′-F and the sugar moiety of each of the remaining nucleotides ofthe antisense strand modified with a modification selected from thegroup consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl,2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety of each of the nucleotides atpositions 2, 3, 4, 5, 7, 8, 10, 14, 16, and 19 of the antisense strandmodified with 2′-F and the sugar moiety of each of the remainingnucleotides of the antisense strand modified with a modificationselected from the group consisting of 2′-O-propargyl, 2′-O-propylamin,2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe),2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl](2′-O-NMA), and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety at position 1, position 2,position 3, position 4, position 5, position 6, position 7, position 8,position 9, position 10, position 11, position 12, position 13, position14, position 15, position 16, position 17, position 18, position 19,position 20, position 21, or position 22 modified with 2′-F.

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety at position 1, position 2,position 3, position 4, position 5, position 6, position 7, position 8,position 9, position 10, position 11, position 12, position 13, position14, position 15, position 16, position 17, position 18, position 19,position 20, position 21, or position 22 modified with 2′-OMe.

In some embodiments, an oligonucleotide provided herein comprises anantisense strand having the sugar moiety at position 1, position 2,position 3, position 4, position 5, position 6, position 7, position 8,position 9, position 10, position 11, position 12, position 13, position14, position 15, position 16, position 17, position 18, position 19,position 20, position 21, or position 22 modified with a modificationselected from the group consisting of 2′-O-propargyl, 2′-O-propylamin,2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe),2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl](2′-O-NMA), and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises asense strand having the sugar moiety at positions 8-11 modified with2′-F. In some embodiments, an oligonucleotide provided herein comprisesa sense strand having the sugar moiety at positions 3, 8, 9, 10, 12, 13and 17 modified with 2′-F. In some embodiments, an oligonucleotideprovided herein comprises a sense strand having the sugar moiety atpositions 1-7 and 12-17 or 12-20 modified with 2′OMe. In someembodiments, an oligonucleotide provided herein comprises a sense strandhaving the sugar moiety of each of the nucleotides at positions 1-7 and12-17 or 12-20 of the sense strand modified with a modification selectedfrom the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino,2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl(2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). In someembodiments, an oligonucleotide provided herein comprises a sense strandhaving the sugar moiety at positions 1-2, 4-7, 11, 14-16, and 18-20modified with 2′OMe. In some embodiments, an oligonucleotide providedherein comprises a sense strand having the sugar moiety of each of thenucleotides at positions 1-2, 4-7, 11, 14-16, and 18-20 of the sensestrand modified with a modification selected from the group consistingof 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl(EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE),2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

In some embodiments, an oligonucleotide provided herein comprises asense strand having the sugar moiety at position 1, position 2, position3, position 4, position 5, position 6, position 7, position 8, position9, position 10, position 11, position 12, position 13, position 14,position 15, position 16, position 17, position 18, position 19,position 20, position 21, position 22, position 23, position 24,position 25, position 26, position 27, position 28, position 29,position 30, position 31, position 32, position 33, position 34,position 35, or position 36 modified with 2′-F.

In some embodiments, an oligonucleotide provided herein comprises asense strand having the sugar moiety at position 1, position 2, position3, position 4, position 5, position 6, position 7, position 8, position9, position 10, position 11, position 12, position 13, position 14,position 15, position 16, position 17, position 18, position 19,position 20, position 21, position 22, position 23, position 24,position 25, position 26, position 27, position 28, position 29,position 30, position 31, position 32, position 33, position 34,position 35, or position 36 modified with 2′-OMe.

In some embodiments, an oligonucleotide provided herein comprises asense strand having the sugar moiety at position 1, position 2, position3, position 4, position 5, position 6, position 7, position 8, position9, position 10, position 11, position 12, position 13, position 14,position 15, position 16, position 17, position 18, position 19,position 20, position 21, position 22, position 23, position 24,position 25, position 26, position 27, position 28, position 29,position 30, position 31, position 32, position 33, position 34,position 35, or position 36 modified with a modification selected fromthe group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino,2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl(2′-MOE), 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA).

5′-Terminal Phosphate

In some embodiments, an oligonucleotide described herein (e.g., an RNAioligonucleotide) comprises a sense strand and an antisense strand,wherein the antisense strand comprises a 5′-terminal phosphate. In someembodiments, 5′-terminal phosphate groups of an RNAi oligonucleotideenhance the interaction with Ago2. However, oligonucleotides comprisinga 5′-phosphate group may be susceptible to degradation via phosphatasesor other enzymes, which can limit their performance and/orbioavailability in vivo. In some embodiments, an oligonucleotide hereinincludes analogs of 5′ phosphates that are resistant to suchdegradation. In some embodiments, the phosphate analog isoxymethylphosphonate, vinylphosphonate or malonylphosphonate, or acombination thereof. In certain embodiments, the 5′ terminus of anoligonucleotide strand is attached to chemical moiety that mimics theelectrostatic and steric properties of a natural 5′-phosphate group(“phosphate mimic”). In some embodiments, the sense and antisensestrands of an oligonucleotide comprise nucleotides sequences selectedfrom the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises a 5′-terminal phosphate,optionally a 5′-terminal phosphate analog.

In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises a 5′-terminal phosphate,optionally a 5′-terminal phosphate analog.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) has a phosphate analog at a 4′-carbon position of thesugar (referred to as a “4′-phosphate analog”). See, e.g., Intl. PatentApplication Publication No. WO 2018/045317. In some embodiments, anoligonucleotide herein comprises a 4′-phosphate analog at a 5′-terminalnucleotide. In some embodiments, a phosphate analog is anoxymethylphosphonate, in which the oxygen atom of the oxymethyl group isbound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. Inother embodiments, a 4′-phosphate analog is a thiomethylphosphonate oran aminomethylphosphonate, in which the sulfur atom of the thiomethylgroup or the nitrogen atom of the amino methyl group is bound to the4′-carbon of the sugar moiety or analog thereof. In certain embodiments,a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments,an oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂,—O—CH₂—PO(OR)₂, or —O—CH₂—PO(OH)(R), in which R is independentlyselected from —H, —CH₃, an alkyl group, —CH₂CH₂CN, —CH₂OCOC(CH₃)₃,—CH₂OCH₂CH₂Si(CH₃)₃ or a protecting group. In certain embodiments, thealkyl group is —CH₂CH₃. More typically, R is independently selected fromH, —CH₃ or —CH₂CH₃. In some embodiment, R is —CH₃. In some embodiments,the 4′-phosphate analog is 5′-methoxyphosphonate-4′-oxy.

In some embodiments, an oligonucleotide provided herein comprises anantisense strand comprising a 4′-phosphate analog at the 5′-terminalnucleotide, wherein 5′-terminal nucleotide comprises the followingstructure (Chem. formula 1):

5′-methoxyphosphonate-4′-oxy-2′-O-methyluridine phosphorothioate[MePhosphonate-4O-mUs] Modified Internucleotide Linkage

In some embodiments, an oligonucleotide provided herein (e.g., a RNAioligonucleotide) comprises a modified internucleotide linkage. In someembodiments, phosphate modifications or substitutions result in anoligonucleotide that comprises at least about 1 (e.g., at least 1, atleast 2, at least 3, or at least 5) modified internucleotide linkage. Insome embodiments, any one of the oligonucleotides disclosed hereincomprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide linkages. Insome embodiments, any one of the oligonucleotides disclosed hereincomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotidelinkages.

A modified internucleotide linkage may be a phosphorodithioate linkage,a phosphorothioate linkage, a phosphotriester linkage, athionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, aphosphoramidite linkage, a phosphonate linkage or a boranophosphatelinkage. In some embodiments, at least one modified internucleotidelinkage of any one of the oligonucleotides as disclosed herein is aphosphorothioate linkage.

In some embodiments, an oligonucleotide provided herein (e.g., a RNAioligonucleotide) has a phosphorothioate linkage between one or more ofpositions 1 and 2 of the sense strand, positions 1 and 2 of theantisense strand, positions 2 and 3 of the antisense strand, positions 3and 4 of the antisense strand, positions 20 and 21 of the antisensestrand, and positions 21 and 22 of the antisense strand. In someembodiments, the oligonucleotide described herein has a phosphorothioatelinkage between each of positions 1 and 2 of the sense strand, positions1 and 2 of the antisense strand, positions 2 and 3 of the antisensestrand, positions 20 and 21 of the antisense strand, and positions 21and 22 of the antisense strand. In some embodiments, the sense andantisense strands of an oligonucleotide comprise nucleotides sequencesselected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises a modified internucleotidelinkage.

In some embodiments, the oligonucleotide described herein has aphosphorothioate linkage between each of positions 1 and 2 of the sensestrand, positions 1 and 2 of the antisense strand, positions 2 and 3 ofthe antisense strand, positions 20 and 21 of the antisense strand, andpositions 21 and 22 of the antisense strand. In some embodiments, thesense and antisense strands of an oligonucleotide comprise nucleotidessequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively, and;(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises a modified internucleotidelinkage.

Base Modifications

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotides) comprises one or more modified nucleobases. In someembodiments, modified nucleobases (also referred to herein as baseanalogs) are linked at the 1′ position of a nucleotide sugar moiety. Incertain embodiments, a modified nucleobase is a nitrogenous base. Insome embodiments, a modified nucleobase does not contain nitrogen atom.See, e.g., US Patent Application Publication No. 2008/0274462. In someembodiments, a modified nucleotide comprises a universal base. In someembodiments, a modified nucleotide does not contain a nucleobase(abasic). In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises one or more modified nucleobases.

In some embodiments, a modified nucleotide comprises a universal base.In some embodiments, a modified nucleotide does not contain a nucleobase(abasic). In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises one or more modified nucleobases.

In some embodiments, a universal base is a heterocyclic moiety locatedat the 1′ position of a nucleotide sugar moiety in a modifiednucleotide, or the equivalent position in a nucleotide sugar moietysubstitution, that, when present in a duplex, can be positioned oppositemore than one type of base without substantially altering structure ofthe duplex. In some embodiments, compared to a reference single-strandednucleic acid (e.g., oligonucleotide) that is fully complementary to atarget nucleic acid (e.g., a NR1H3 mRNA), a single-stranded nucleic acidcontaining a universal base forms a duplex with the target nucleic acidthat has a lower T_(m) than a duplex formed with the complementarynucleic acid. In some embodiments, when compared to a referencesingle-stranded nucleic acid in which the universal base has beenreplaced with a base to generate a single mismatch, the single-strandednucleic acid containing the universal base forms a duplex with thetarget nucleic acid that has a higher T_(m) than a duplex formed withthe nucleic acid comprising the mismatched base.

Non-limiting examples of universal-binding nucleotides include, but arenot limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole and/or1-β-D-ribofuranosyl-3-nitropyrrole (see, US Patent ApplicationPublication No. 2007/0254362; Van Aerschot et al. NUCLEIC ACIDS RES.(1995); 23: 4363-4370; Loakes et al. NUCLEIC ACIDS RES. (1995); 23:2361-66; and Loakes & Brown NUCLEIC ACIDS RES. (1994); 22: 4039-43).

Targeting Ligands

In some embodiments, it is desirable to target an oligonucleotideprovided herein (e.g., an RNAi oligonucleotide) to one or more cells orcell type, tissues, organs, or anatomical regions or compartments. Sucha strategy may help to avoid undesirable effects and/or to avoid undueloss of the oligonucleotide to cells, tissues, organs, or anatomicalregions or compartments that would not benefit from the oligonucleotideor its effects (e.g., inhibition or reduction of NR1H3 expression).Accordingly, in some embodiments, oligonucleotides disclosed herein(e.g., RNAi oligonucleotides) are modified to facilitate targetingand/or delivery to particular cells or cell types, tissues, organs, oranatomical regions or compartments (e.g., to facilitate delivery of theoligonucleotide to the liver). In some embodiments, an oligonucleotidecomprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or morenucleotides) conjugated to one or more targeting ligand(s). In someembodiments, the sense and antisense strands of an oligonucleotidecomprise nucleotides sequences selected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises a targeting ligand conjugated toat least one nucleotide.

In some embodiments, an oligonucleotide comprises at least onenucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) conjugated toone or more targeting ligand(s). In some embodiments, the sense andantisense strands of an oligonucleotide comprise nucleotides sequencesselected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively, and;(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises a targeting ligand conjugated toat least one nucleotide.

In some embodiments, the targeting ligand comprises a carbohydrate,amino sugar, cholesterol, peptide, polypeptide, protein, or part of aprotein (e.g., an antibody or antibody fragment), or lipid. In certainembodiments, the targeting ligand is a carbohydrate comprising at leastone GalNAc moiety.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotidesof an oligonucleotide provided herein (e.g., an RNAi oligonucleotide)are each conjugated to a separate targeting ligand (e.g., a GalNAcmoiety). In some embodiments, 2 to 4 nucleotides of an oligonucleotideare each conjugated to a separate targeting ligand. In some embodiments,targeting ligands are conjugated to 2 to 4 nucleotides at either ends ofthe sense or antisense strand (e.g., targeting ligands are conjugated toa 2 to 4 nucleotide overhang or extension on the 5′ or 3′ terminus ofthe sense or antisense strand) such that the targeting ligands resemblebristles of a toothbrush and the oligonucleotide resembles a toothbrush.For example, an oligonucleotide may comprise a stem-loop at either the5′ or 3′ terminus of the sense strand and 1, 2, 3, or 4 nucleotides ofthe loop of the stem may be individually conjugated to a targetingligand. In some embodiments, an oligonucleotide provided by thedisclosure (e.g., a RNAi oligonucleotide) comprises a stem-loop at the3′ terminus of the sense strand, wherein the loop of the stem-loopcomprises a triloop or a tetraloop, and wherein the 3 or 4 nucleotidescomprising the triloop or tetraloop, respectively, are individuallyconjugated to a targeting ligand. In some embodiments, anoligonucleotide provided by the disclosure (e.g., a RNAioligonucleotide) comprises a stem-loop at the 3′ terminus of the sensestrand, wherein the loop of the stem-loop comprises a tetraloop, andwherein 3 nucleotides of the tetraloop are individually conjugated to atargeting ligand.

GalNAc is a high affinity carbohydrate ligand for the asialoglycoproteinreceptor (ASGPR), which is primarily expressed on the surface ofhepatocyte cells and has a major role in binding, internalizing andsubsequent clearing circulating glycoproteins that contain terminalgalactose or GalNAc residues (asialoglycoproteins). Conjugation (eitherindirect or direct) of GalNAc moieties to oligonucleotides of theinstant disclosure can be used to target these oligonucleotides to theASGPR expressed on cells. In some embodiments, an oligonucleotide of theinstant disclosure (e.g., an RNAi oligonucleotide) is conjugated to atleast one or more GalNAc moieties, wherein the GalNAc moieties targetthe oligonucleotide to an ASGPR expressed on human liver cells (e.g.,human hepatocytes). In some embodiments, the GalNAc moiety target theoligonucleotide to the liver.

In some embodiments, an oligonucleotide of the instant disclosure (e.g.,an RNAi oligonucleotide) is conjugated directly or indirectly to amonovalent GalNAc moiety. In some embodiments, the oligonucleotide isconjugated directly or indirectly to more than one monovalent GalNAc(i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties and istypically conjugated to 3 or 4 monovalent GalNAc moieties). In someembodiments, an oligonucleotide is conjugated to one or more bivalentGalNAc, trivalent GalNAc or tetravalent GalNAc moieties. In someembodiments, a bivalent, trivalent or tetravalent GalNAc moiety isconjugated to an oligonucleotide via a branched linker. In someembodiments, a monovalent GalNAc moiety is conjugated to a firstnucleotide and a bivalent, trivalent, or tetravalent GalNAc moiety isconjugated to a second nucleotide via a branched linker.

In some embodiments, one (1) or more (e.g., 1, 2, 3, 4, 5, or 6)nucleotides of an oligonucleotide described herein (e.g., an RNAioligonucleotide) are each conjugated to a GalNAc moiety. In someembodiments, two (2) to four (4) nucleotides of a tetraloop are eachconjugated to a separate GalNAc moiety. In some embodiments, one (1) tothree (3) nucleotides of a triloop are each conjugated to a separateGalNAc moiety. In some embodiments, targeting ligands are conjugated totwo (2) to four (4) nucleotides at either ends of the sense or antisensestrand (e.g., ligands are conjugated to a two (2) to four (4) nucleotideoverhang or extension on the 5′ or 3′ terminus of the sense or antisensestrand) such that the GalNAc moieties resemble bristles of a toothbrushand the oligonucleotide resembles a toothbrush. In some embodiments,GalNAc moieties are conjugated to a nucleotide of the sense strand. Forexample, three (3) or four (4) GalNAc moieties can be conjugated tonucleotides in the tetraloop of the sense strand where each GalNAcmoiety is conjugated to one (1) nucleotide.

In some embodiments, an oligonucleotide described herein (e.g., an RNAioligonucleotide) comprises a tetraloop, wherein the tetraloop (L) is anycombination of adenine (A) and guanine (G) nucleotides. In someembodiments, the tetraloop (L) comprises a monovalent GalNAc moietyattached to any one or more guanine (G) nucleotides of the tetraloop viaany linker described herein, as depicted below (X=heteroatom) in Chem.formula 2:

In some embodiments, the tetraloop (L) has a monovalent GalNAc attachedto any one or more adenine nucleotides of the tetraloop via any linkerdescribed herein, as depicted below (X=heteroatom) in Chem. formula 3:

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide) comprises a monovalent GalNAc moiety attached to aguanine (G) nucleotide referred to as [ademG-GalNAc] or2′-aminodiethoxymethanol-Guanine-GalNAc, as depicted below in Chem.formula 4:

In some embodiments, an oligonucleotide herein comprises a monovalentGalNAc moiety attached to an adenine nucleotide, referred to as[ademA-GalNAc] or 2′-aminodiethoxymethanol-Adenine-GalNAc, as depictedbelow Chem. formula 5.

An example of such conjugation is shown below for a loop comprising from5′ to 3′ the nucleotide sequence GAAA (L=linker, X=heteroatom). Such aloop may be present, for example, at positions 27-30 of a sense strandprovided herein. In the Chem. formula 6 is used to describe anattachment point to the oligonucleotide strand.

Appropriate methods or chemistry (e.g., click chemistry) can be used tolink a targeting ligand to a nucleotide. In some embodiments, atargeting ligand is conjugated to a nucleotide comprising anoligonucleotide herein (e.g., an RNAi oligonucleotide) using a clicklinker. In some embodiments, an acetal-based linker is used to conjugatea targeting ligand to a nucleotide of any one of the oligonucleotidesdescribed herein. Acetal-based linkers are disclosed, for example, inIntl. Patent Application Publication No. WO2016/100401. In someembodiments, the linker is a labile linker. However, in otherembodiments, the linker is stable. An example is shown below for a loopcomprising from 5′ to 3′ the nucleotides GAAA, in which GalNAc moietiesare attached to nucleotides of the loop using an acetal linker. Such aloop may be present, for example, at positions 27-30 of the any one ofthe sense strands. In the Chem. formula 7 is an attachment point to theoligonucleotide strand.

As mentioned, various appropriate methods or chemistry synthetictechniques (e.g., click chemistry) can be used to link a targetingligand to a nucleotide. In some embodiments, a targeting ligand isconjugated to a nucleotide using a click linker. In some embodiments, anacetal-based linker is used to conjugate a targeting ligand to anucleotide of any one of the oligonucleotides described herein.Acetal-based linkers are disclosed, for example, in Intl. PatentApplication Publication No. WO 2016/100401. In some embodiments, thelinker is a labile linker. However, in other embodiments, the linker isa stable linker.

In some embodiments, a duplex extension (e.g., of up to 3, 4, 5, or 6 bpin length) is provided between a targeting ligand (e.g., a GalNAcmoiety) and the oligonucleotide. In some embodiments, theoligonucleotides herein (e.g., RNAi oligonucleotides) do not have aGalNAc conjugated thereto.

In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,wherein the oligonucleotide comprises at least one GalNAc moietyconjugated to a nucleotide.

In some embodiments, the sense and antisense strands of anoligonucleotide comprise nucleotides sequences selected from the groupconsisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively,wherein the oligonucleotide comprises at least one GalNAc moietyconjugated to a nucleotide.

Exemplary Oligonucleotides for Reducing NR1H3 Expression

In some embodiments, the NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression provided by the disclosure comprise a sensestrand and an antisense strand, wherein all nucleotides comprising thesense strand and antisense strand are modified, wherein the antisensestrand comprises a region of complementarity to a NR1H3 mRNA targetsequence of any one of SEQ ID NOs: 1-384, and wherein the region ofcomplementarity is at least 15 contiguous nucleotides in length. In someembodiments, the 5′-terminal nucleotide of the antisense strandcomprises 5′-methoxyphosphonate-4′-oxy-2′-O-methyluridine[MePhosphonate-4O-mU], as described herein. In some embodiments, the5′-terminal nucleotide of the antisense strand comprises aphosphorothioate linkage. In some embodiments, the antisense strand andthe sense strand comprise one or more 2′-fluoro (2′-F) and 2′-O-methyl(2′-OMe) modified nucleotides and at least one phosphorothioate linkage.In some embodiments, the antisense strand comprises four (4)phosphorothioate linkages and the sense strand comprises one (1)phosphorothioate linkage. In some embodiments, the antisense strandcomprises five (5) phosphorothioate linkages and the sense strandcomprises one (1) phosphorothioate linkage.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand having a sequence of any oneof SEQ ID NOs: 1-384 and an antisense strand comprising a complementarysequence selected from SEQ ID NOs: 385-768.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand having a sequence of any oneof SEQ ID NOs: 769-856 and an antisense strand comprising acomplementary sequence selected from SEQ ID NOs: 857-944.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand having a sequence of any oneof SEQ ID NOs: 1519-1552 and an antisense strand comprising acomplementary sequence selected from SEQ ID NOs: 857-944.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) comprises a sense strand having a sequence of any oneof SEQ ID NOs: 945-1032 and an antisense strand comprising acomplementary sequence selected from SEQ ID NOs: 1033-1120.

In some embodiments, an oligonucleotide provided herein (e.g., and RNAioligonucleotide) for reducing NR1H3 expression comprises:

a sense strand comprising a 2′-F modified nucleotide at positions 8-11,a 2′-OMe modified nucleotide at positions 1-7, 12-27, and 31-36, aGalNAc-conjugated nucleotide at position 28, 29 and 30; and aphosphorothioate linkage between positions 1 and 2;

an antisense strand comprising a 2′-F modified nucleotide at positions2, 3, 4, 5, 7, 10 and 14, a 2′-OMe at positions 1, 6, 8, 9, 11-13, and15-22, a phosphorothioate linkage between positions 1 and 2, positions 2and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22,and a 5′-terminal nucleotide at position 1 comprising a 4′-phosphateanalog, optionally wherein the 5′-terminal nucleotide comprises5′-methoxyphosphonate-4′-oxy-2′-O-methyluridine [MePhosphonate-4O-mU];wherein positions 1-20 of the antisense strand form a duplex region withpositions 1-20 of the sense strand, wherein positions 21-36 of the sensestrand form a stem-loop, wherein positions 27-30 form the loop of thestem-loop, optionally wherein positions 27-30 comprise a tetraloop,wherein positions 21 and 22 of the antisense strand comprise anoverhang, and wherein the sense strand and antisense strands comprisenucleotide sequences selected from the group consisting of:

(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively,

In some embodiments, the NR1H3-targeting dsRNAi oligonucleotides forreducing NR1H3 expression comprise:

a sense strand comprising a 2′-F modified nucleotide at positions 8-11,a 2′-OMe modified nucleotide at positions 1-7, 12-27, and 31-36, aGalNAc-conjugated nucleotide at position 28, 29 and 30; and aphosphorothioate linkage between positions 1 and 2;

an antisense strand comprising a 2′-F modified nucleotide at positions2, 3, 4, 5, 7, 10 and 14, a 2′-OMe at positions 1, 6, 8, 9, 11-13, and15-22, a phosphorothioate linkage between positions 1 and 2, positions 2and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22,and a 5′-terminal nucleotide at position 1 comprising a 4′-phosphateanalog, optionally wherein the 5′-terminal nucleotide comprises5′-methoxyphosphonate-4′-oxy-2′-O-methyluridine [MePhosphonate-4O-mU];wherein positions 1-20 of the antisense strand form a duplex region withpositions 1-20 of the sense strand, wherein positions 21-36 of the sensestrand form a stem-loop, wherein positions 27-30 form the loop of thestem-loop, optionally wherein positions 27-30 comprise a tetraloop,wherein positions 21 and 22 of the antisense strand comprise anoverhang, and wherein the sense strand and antisense strands comprisenucleotide sequences selected from the group consisting of:

(a) SEQ ID NOs: 786 and 874, respectively;(b) SEQ ID NOs: 787 and 875, respectively;(c) SEQ ID NOs: 1537 and 929, respectively; and,(d) SEQ ID NOs: 813 and 901, respectively.

In some embodiments, a NR1H3-targeting oligonucleotide for reducingNR1H3 expression provided by the disclosure comprises a sense strandcomprising the nucleotide sequence as set forth in SEQ ID NO: 786 and anantisense strand comprising the nucleotide sequence as set forth in SEQID NO: 874. In some embodiments, a NR1H3-targeting oligonucleotide forreducing NR1H3 expression provided by the disclosure comprises a sensestrand comprising the nucleotide sequence as set forth in SEQ ID NO: 787and an antisense strand comprising the nucleotide sequence as set forthin SEQ ID NO: 875. In some embodiments, a NR1H3-targetingoligonucleotide for reducing NR1H3 expression provided by the disclosurecomprises a sense strand comprising the nucleotide sequence as set forthin SEQ ID NO: 1537 and an antisense strand comprising the nucleotidesequence as set forth in SEQ ID NO: 929. In some embodiments, aNR1H3-targeting oligonucleotide for reducing NR1H3 expression providedby the disclosure comprises a sense strand comprising the nucleotidesequence as set forth in SEQ ID NO: 813 and an antisense strandcomprising the nucleotide sequence as set forth in SEQ ID NO: 901.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1512; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the antisense and sense strands are separate strands which forman asymmetric duplex region having an overhang of 1-4 nucleotides at the3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1513; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the antisense and sense strands are separate strands which forman asymmetric duplex region having an overhang of 1-4 nucleotides at the3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1514; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the antisense and sense strands are separate strands which forman asymmetric duplex region having an overhang of 1-4 nucleotides at the3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1515; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the antisense and sense strands are separate strands which forman asymmetric duplex region having an overhang of 1-4 nucleotides at the3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1512; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the stem-loop is set forth asS1-L-S2, wherein S1 is complementary to S2 and wherein L forms a loopbetween S1 and S2 of 3 to 5 nucleotides in length, wherein the antisenseand sense strands are separate strands which form an asymmetric duplexregion having an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1513; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the stem-loop is set forth asS1-L-S2, wherein S1 is complementary to S2 and wherein L forms a loopbetween S1 and S2 of 3 to 5 nucleotides in length, wherein the antisenseand sense strands are separate strands which form an asymmetric duplexregion having an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1514; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the stem-loop is set forth asS1-L-S2, wherein S1 is complementary to S2 and wherein L forms a loopbetween S1 and S2 of 3 to 5 nucleotides in length, wherein the antisenseand sense strands are separate strands which form an asymmetric duplexregion having an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1515; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the stem-loop is set forth asS1-L-S2, wherein S1 is complementary to S2 and wherein L forms a loopbetween S1 and S2 of 3 to 5 nucleotides in length, wherein the antisenseand sense strands are separate strands which form an asymmetric duplexregion having an overhang of 1-4 nucleotides at the 3′ terminus of theantisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1512; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the region of complementarity to the antisense strand is setforth in SEQ ID NO: 1509, wherein the antisense and sense strands areseparate strands which form an asymmetric duplex region having anoverhang of 1-4 nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1513; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the region of complementarity to the antisense strand is setforth in SEQ ID NO: 1510, wherein the antisense and sense strands areseparate strands which form an asymmetric duplex region having anoverhang of 1-4 nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1514; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the region of complementarity to the antisense strand is setforth in SEQ ID NO: 1409, wherein the antisense and sense strands areseparate strands which form an asymmetric duplex region having anoverhang of 1-4 nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1515; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strand,wherein the region of complementarity to the antisense strand is setforth in SEQ ID NO: 1511, wherein the antisense and sense strands areseparate strands which form an asymmetric duplex region having anoverhang of 1-4 nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1512; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the region of complementarityto the antisense strand is set forth in SEQ ID NO: 1509, wherein thestem-loop is set forth as S1-L-S2, wherein S1 is complementary to S2 andwherein L forms a loop between S1 and S2 of 3 to 5 nucleotides inlength, wherein the antisense and sense strands are separate strandswhich form an asymmetric duplex region having an overhang of 1-4nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1513; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the region of complementarityto the antisense strand is set forth in SEQ ID NO: 1510, wherein thestem-loop is set forth as S1-L-S2, wherein S1 is complementary to S2 andwherein L forms a loop between S1 and S2 of 3 to 5 nucleotides inlength, wherein the antisense and sense strands are separate strandswhich form an asymmetric duplex region having an overhang of 1-4nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1514; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the region of complementarityto the antisense strand is set forth in SEQ ID NO: 1409, wherein thestem-loop is set forth as S1-L-S2, wherein S1 is complementary to S2 andwherein L forms a loop between S1 and S2 of 3 to 5 nucleotides inlength, wherein the antisense and sense strands are separate strandswhich form an asymmetric duplex region having an overhang of 1-4nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, a NR1H3-targeting dsRNAi oligonucleotide forreducing NR1H3 expression comprises (i) an antisense strand of 19-30nucleotides in length, wherein the antisense strand comprises anucleotide sequence comprising a region of complementarity to a NR1H3mRNA target sequence, wherein the region of complementarity is set forthin SEQ ID NO: 1515; and (ii) a sense strand of 19-50 nucleotides inlength comprising a region of complementarity to the antisense strandand a stem-loop at the 3′terminus, wherein the region of complementarityto the antisense strand is set forth in SEQ ID NO: 1511, wherein thestem-loop is set forth as S1-L-S2, wherein S1 is complementary to S2 andwherein L forms a loop between S1 and S2 of 3 to 5 nucleotides inlength, wherein the antisense and sense strands are separate strandswhich form an asymmetric duplex region having an overhang of 1-4nucleotides at the 3′ terminus of the antisense strand.

In some embodiments, the disclosure provides an oligonucleotide (e.g.,an RNAi oligonucleotide) for reducing NR1H3 expression, wherein theoligonucleotide comprises a sense strand and an antisense strandaccording to:

Sense Strand: 5′-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-[adem A-GalNAc]-[adem A-GalNAc]-[adem A-GalNAc]-mX-mX-mX- mX-mX-mX-3';hybridized to:

Antisense Strand: 5′-[MePhosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S- mX-3′;wherein mX=2′-O-methyl modified nucleotide, fX=2′-fluoro modifiednucleotide, —S—=phosphorothioate linkage, -=phosphodiester linkage,[MePhosphonate-4O-mX]=5′-methoxyphosphonate-4-oxy modified nucleotide,and ademA-GalNAc=GalNAc attached to an adenine nucleotide.

In some embodiments, the disclosure provides an oligonucleotide (e.g.,an RNAi oligonucleotide) for reducing NR1H3 expression, wherein theoligonucleotide comprises a sense strand and an antisense strandaccording to:

Sense Strand: 5′-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-[adem A-GalNAc]-[adem A-GalNAc]-[adem A-GalNAc]- mX-mX-mX-mX-mX-mX-3′;hybridized to:

Antisense Strand: 5′-[MePhosphonate-4O-mX]-S-fX-S-fX-S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX- S-mX-S-mX-3′;wherein mX=2′-O-methyl modified nucleotide, fX=2′-fluoro modifiednucleotide, —S—=phosphorothioate linkage, -=phosphodiester linkage,[MePhosphonate-4O-mX]=5′-methoxyphosphonate-4-oxy modified nucleotide,and ademA-GalNAc=GalNAc attached to an adenine nucleotide.

In some embodiments, the disclosure provides an oligonucleotide (e.g.,an RNAi oligonucleotide) for reducing NR1H3 expression, wherein theoligonucleotide comprises a sense strand and an antisense strandcomprising nucleotide sequences selected from the group consisting of:

(a) SEQ ID NOs: 945 and 1033, respectively;(b) SEQ ID NOs: 946 and 1034, respectively;(c) SEQ ID NOs: 947 and 1035, respectively;(d) SEQ ID NOs: 948 and 1036, respectively;(e) SEQ ID NOs: 949 and 1037, respectively;(f) SEQ ID NOs: 950 and 1038, respectively;(g) SEQ ID NOs: 951 and 1039, respectively;(h) SEQ ID NOs: 952 and 1040, respectively;(i) SEQ ID NOs: 953 and 1041, respectively;(j) SEQ ID NOs: 954 and 1042, respectively;(k) SEQ ID NOs: 955 and 1043, respectively;(l) SEQ ID NOs: 956 and 1044 respectively;(m) SEQ ID NOs: 957 and 1045, respectively;(n) SEQ ID NOs: 958 and 1046, respectively;(o) SEQ ID NOs: 959 and 1047, respectively;(p) SEQ ID NOs: 960 and 1048, respectively;(q) SEQ ID NOs: 961 and 1049, respectively;(r) SEQ ID NOs: 962 and 1050, respectively;(s) SEQ ID NOs: 963 and 1051, respectively;(t) SEQ ID NOs: 964 and 1052, respectively;(u) SEQ ID NOs: 965 and 1053, respectively;(v) SEQ ID NOs: 966 and 1054, respectively;(w) SEQ ID NOs: 967 and 1055, respectively;(x) SEQ ID NOs: 968 and 1056, respectively;(y) SEQ ID NOs: 969 and 1057, respectively;(z) SEQ ID NOs: 970 and 1058, respectively;(aa) SEQ ID NOs: 971 and 1059, respectively;(bb) SEQ ID NOs: 972 and 1060, respectively;(cc) SEQ ID NOs: 973 and 1061, respectively;(dd) SEQ ID NOs: 974 and 1062, respectively;(ee) SEQ ID NOs: 975 and 1063, respectively;(ff) SEQ ID NOs: 976 and 1064, respectively;(gg) SEQ ID NOs: 977 and 1065, respectively;(hh) SEQ ID NOs: 978 and 1066, respectively;(ii) SEQ ID NOs: 979 and 1067, respectively;(jj) SEQ ID NOs: 980 and 1068, respectively;(kk) SEQ ID NOs: 981 and 1069, respectively;(ll) SEQ ID NOs: 982 and 1070, respectively;(mm) SEQ ID NOs: 983 and 1071, respectively;(nn) SEQ ID NOs: 984 and 1072, respectively;(oo) SEQ ID NOs: 985 and 1073, respectively;(pp) SEQ ID NOs: 986 and 1074, respectively;(qq) SEQ ID NOs: 987 and 1075, respectively;(rr) SEQ ID NOs: 988 and 1076, respectively;(ss) SEQ ID NOs: 989 and 1077, respectively;(tt) SEQ ID NOs: 990 and 1078, respectively;(uu) SEQ ID NOs: 991 and 1079, respectively;(vv) SEQ ID NOs: 992 and 1080, respectively;(ww) SEQ ID NOs: 993 and 1081, respectively;(xx) SEQ ID NOs: 994 and 1082, respectively;(yy) SEQ ID NOs: 995 and 1083, respectively;(zz) SEQ ID NOs: 996 and 1084, respectively;(aaa) SEQ ID NOs: 997 and 1085, respectively;(bbb) SEQ ID NOs: 998 and 1086, respectively;(ccc) SEQ ID NOs: 999 and 1087, respectively;(ddd) SEQ ID NOs: 1000 and 1088, respectively;(eee) SEQ ID NOs: 1001 and 1089, respectively;(fff) SEQ ID NOs: 1002 and 1090, respectively;(ggg) SEQ ID NOs: 1003 and 1091, respectively;(hhh) SEQ ID NOs: 1004 and 1092 respectively;(iii) SEQ ID NOs: 1005 and 1093 respectively;(jjj) SEQ ID NOs: 1006 and 1094, respectively;(kkk) SEQ ID NOs: 1007 and 1095, respectively;(lll) SEQ ID NOs: 1008 and 1096, respectively;(mmm) SEQ ID NOs: 1009 and 1097, respectively;(nnn) SEQ ID NOs: 1010 and 1098, respectively;(ooo) SEQ ID NOs: 1011 and 1099, respectively;(ppp) SEQ ID NOs: 1012 and 1100, respectively;(qqq) SEQ ID NOs: 1013 and 1101, respectively;(rrr) SEQ ID NOs: 1014 and 1102 respectively;(sss) SEQ ID NOs: 1015 and 1103, respectively;(ttt) SEQ ID NOs: 1016 and 1104, respectively;(uuu) SEQ ID NOs: 1017 and 1105, respectively;(vvv) SEQ ID NOs: 1018 and 1106, respectively;(www) SEQ ID NOs: 1019 and 1107, respectively;(xxx) SEQ ID NOs: 1020 and 1108, respectively;(yyy) SEQ ID NOs: 1021 and 1109, respectively;(zzz) SEQ ID NOs: 1022 and 1110, respectively;(aaaa) SEQ ID NOs: 1023 and 1111, respectively;(bbbb) SEQ ID NOs: 1024 and 1112, respectively;(cccc) SEQ ID NOs: 1025 and 1113, respectively;(dddd) SEQ ID NOs: 1026 and 1114, respectively;(eeee) SEQ ID NOs: 1027 and 1115, respectively;(ffff) SEQ ID NOs: 1028 and 1116, respectively;(gggg) SEQ ID NOs: 1029 and 1117, respectively;(hhhh) SEQ ID NOs: 1030 and 1118, respectively;(iiii) SEQ ID NOs: 1031 and 1119, respectively; and,(jjjj) SEQ ID NOs: 1032 and 1120, respectively.

In some embodiments, a NR1H3-targeting oligonucleotide for reducingNR1H3 expression provided by the disclosure comprises a sense strandcomprising the nucleotide sequence as set forth in SEQ ID NO: 963 and anantisense strand comprising the nucleotide sequence as set forth in SEQID NO: 1051. In some embodiments, a NR1H3-targeting oligonucleotide forreducing NR1H3 expression provided by the disclosure comprises a sensestrand comprising the nucleotide sequence as set forth in SEQ ID NO: 964and an antisense strand comprising the nucleotide sequence as set forthin SEQ ID NO: 1052. In some embodiments, a NR1H3-targetingoligonucleotide for reducing NR1H3 expression provided by the disclosurecomprises a sense strand comprising the nucleotide sequence as set forthin SEQ ID NO: 1006 and an antisense strand comprising the nucleotidesequence as set forth in SEQ ID NO: 1094. In some embodiments, aNR1H3-targeting oligonucleotide for reducing NR1H3 expression providedby the disclosure comprises a sense strand comprising the nucleotidesequence as set forth in SEQ ID NO: 1018 and an antisense strandcomprising the nucleotide sequence as set forth in SEQ ID NO: 1106.

Formulations

Various formulations (e.g., pharmaceutical formulations) have beendeveloped for oligonucleotide use. For example, oligonucleotides (e.g.,RNAi oligonucleotides) can be delivered to a subject or a cellularenvironment using a formulation that minimizes degradation, facilitatesdelivery and/or uptake, or provides another beneficial property to theoligonucleotides in the formulation. In some embodiments, providedherein are compositions comprising oligonucleotides (e.g., RNAioligonucleotides) reduce the expression of NR1H3. Such compositions canbe suitably formulated such that when administered to a subject, eitherinto the immediate environment of a target cell or systemically, asufficient portion of the oligonucleotides enter the cell to reduceNR1H3 expression. Any variety of suitable oligonucleotide formulationscan be used to deliver oligonucleotides for the reduction of NR1H3 asdisclosed herein. In some embodiments, an oligonucleotide is formulatedin buffer solutions such as phosphate buffered saline solutions,liposomes, micellar structures, and capsids. Any of the oligonucleotidesdescribed herein may be provided not only as nucleic acids, but also inthe form of a pharmaceutically acceptable salt.

Formulations of oligonucleotides with cationic lipids can be used tofacilitate transfection of the oligonucleotides into cells. For example,cationic lipids, such as lipofectin, cationic glycerol derivatives, andpolycationic molecules (e.g., polylysine), can be used. Suitable lipidsinclude Oligofectamine, Lipofectamine (Life Technologies), NC388(Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche)all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, a formulation comprises a lipidnanoparticle. In some embodiments, an excipient comprises a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a subject in need thereof (see, e.g.,Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition,Pharmaceutical Press, 2013).

In some embodiments, the formulations herein comprise an excipient. Insome embodiments, an excipient confers to a composition improvedstability, improved absorption, improved solubility and/or therapeuticenhancement of the active ingredient. In some embodiments, an excipientis a buffering agent (e.g., sodium citrate, sodium phosphate, a trisbase, or sodium hydroxide) or a vehicle (e.g., a buffered solution,petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, anoligonucleotide is lyophilized for extending its shelf-life and thenmade into a solution before use (e.g., administration to a subject).Accordingly, an excipient in a composition comprising any one of theoligonucleotides described herein may be a lyoprotectant (e.g.,mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone) or acollapse temperature modifier (e.g., dextran, Ficoll™ or gelatin).

In some embodiments, a pharmaceutical composition is formulated to becompatible with its intended route of administration. The route ofadministration may be any route which effectively transports a compoundof this invention to the desired or appropriate place in the body.Examples of routes of administration include parenteral (e.g.,intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous),oral (e.g., inhalation), transdermal (e.g., topical), transmucosal andrectal administration.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Sterile injectable solutions can beprepared by incorporating the oligonucleotides in a required amount in aselected solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization.

In some embodiments, a composition may contain at least about 0.1% ofthe therapeutic agent (e.g., a RNAi oligonucleotide for reducing NR1H3expression) or more, although the percentage of the active ingredient(s)may be between about 1% to about 80% or more of the weight or volume ofthe total composition. Factors such as solubility, bioavailability,biological half-life, route of administration, product shelf life, aswell as other pharmacological considerations will be contemplated by oneskilled in the art of preparing such pharmaceutical formulations, and assuch, a variety of dosages and treatment regimens may be desirable.

Methods of Use Reducing NR1H3 Expression

In some embodiments, the disclosure provides methods for contacting ordelivering to a cell or population of cells an effective amount ofoligonucleotides provided herein (e.g., RNAi oligonucleotides) to reduceNR1H3 expression. In some embodiments, a reduction of NR1H3 expressionis determined by measuring a reduction in the amount or level of NR1H3mRNA, NR1H3 protein, or NR1H3 activity in a cell. The methods includethose described herein and known to one of ordinary skill in the art.

Methods provided herein are useful in any appropriate cell type. In someembodiments, a cell is any cell that expresses NR1H3 mRNA (e.g.,hepatocytes). In some embodiments, the cell is a primary cell obtainedfrom a subject. In some embodiments, the primary cell has undergone alimited number of passages such that the cell substantially maintainsits natural phenotypic properties. In some embodiments, a cell to whichthe oligonucleotide is delivered is ex vivo or in vitro (i.e., can bedelivered to a cell in culture or to an organism in which the cellresides).

In some embodiments, the oligonucleotides herein (e.g., RNAioligonucleotides) are delivered to a cell or population of cells using anucleic acid delivery method known in the art including, but not limitedto, injection of a solution containing the oligonucleotides, bombardmentby particles covered by the oligonucleotides, exposing the cell orpopulation of cells to a solution containing the oligonucleotides, orelectroporation of cell membranes in the presence of theoligonucleotides. Other methods known in the art for deliveringoligonucleotides to cells may be used, such as lipid-mediated carriertransport, chemical-mediated transport, and cationic liposometransfection such as calcium phosphate, and others.

In some embodiments, reduction of NR1H3 expression is determined by anassay or technique that evaluates one or more molecules, properties, orcharacteristics of a cell or population of cells associated with NR1H3expression, or by an assay or technique that evaluates molecules thatare directly indicative of NR1H3 expression in a cell or population ofcells (e.g., NR1H3 mRNA or NR1H3 protein). In some embodiments, theextent to which an oligonucleotide provided herein reduces NR1H3expression is evaluated by comparing NR1H3 expression in a cell orpopulation of cells contacted with the oligonucleotide to an appropriatecontrol (e.g., an appropriate cell or population of cells not contactedwith the oligonucleotide or contacted with a control oligonucleotide).In some embodiments, a control amount or level of NR1H3 expression in acontrol cell or population of cells is predetermined, such that thecontrol amount or level need not be measured in every instance the assayor technique is performed. The predetermined level or value can take avariety of forms. In some embodiments, a predetermined level or valuecan be single cut-off value, such as a median or mean.

In some embodiments, contacting or delivering an oligonucleotidedescribed herein (e.g., an RNAi oligonucleotide) to a cell or apopulation of cells results in a reduction in NR1H3 expression in a cellor population of cells not contacted with the oligonucleotide orcontacted with a control oligonucleotide. In some embodiments, thereduction in NR1H3 expression is about 1% or lower, about 5% or lower,about 10% or lower, about 15% or lower, about 20% or lower, about 25% orlower, about 30% or lower, about 35% or lower, about 40% or lower, about45% or lower, about 50% or lower, about 55% or lower, about 60% orlower, about 70% or lower, about 80% or lower, or about 90% or lowerrelative to a control amount or level of NR1H3 expression. In someembodiments, the control amount or level of NR1H3 expression is anamount or level of NR1H3 mRNA and/or NR1H3 protein in a cell orpopulation of cells that has not been contacted with an oligonucleotideherein. In some embodiments, the effect of delivery of anoligonucleotide herein to a cell or population of cells according to amethod herein is assessed after any finite period or amount of time(e.g., minutes, hours, days, weeks, months). For example, in someembodiments, NR1H3 expression is determined in a cell or population ofcells at least about 4 hours, about 8 hours, about 12 hours, about 18hours, about 24 hours; or at least about 1 day, about 2 days, about 3days, about 4 days, about 5 days, about 6 days, about 7 days, about 8days, about 9 days, about 10 days, about 11 days, about 12 days, about13 days, about 14 days, about 21 days, about 28 days, about 35 days,about 42 days, about 49 days, about 56 days, about 63 days, about 70days, about 77 days, or about 84 days or more after contacting ordelivering the oligonucleotide to the cell or population of cells. Insome embodiments, NR1H3 expression is determined in a cell or populationof cells at least about 1 month, about 2 months, about 3 months, about 4months, about 5 months, or about 6 months or more after contacting ordelivering the oligonucleotide to the cell or population of cells.

In some embodiments, an oligonucleotide provided herein (e.g., an RNAioligonucleotide) is delivered in the form of a transgene that isengineered to express in a cell the oligonucleotide or strandscomprising the oligonucleotide (e.g., its sense and antisense strands).In some embodiments, an oligonucleotide herein is delivered using atransgene engineered to express any oligonucleotide disclosed herein.Transgenes may be delivered using viral vectors (e.g., adenovirus,retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpessimplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs).In some embodiments, transgenes can be injected directly to a subject.

Treatment Methods

The disclosure provides oligonucleotides (e.g., RNAi oligonucleotides)for use as a medicament, in particular for use in a method for thetreatment of diseases, disorders, and conditions associated withexpression of NR1H3. The disclosure also provides oligonucleotides foruse, or adaptable for use, to treat a subject (e.g., a human having adisease, disorder or condition associated with NR1H3 expression) thatwould benefit from reducing NR1H3 expression. In some respects, thedisclosure provides oligonucleotides for use, or adapted for use, totreat a subject having a disease, disorder or condition associated withexpression of NR1H3. The disclosure also provides oligonucleotides foruse, or adaptable for use, in the manufacture of a medicament orpharmaceutical composition for treating a disease, disorder or conditionassociated with NR1H3 expression. In some embodiments, theoligonucleotides for use, or adaptable for use, target NR1H3 mRNA andreduce NR1H3 expression (e.g., via the RNAi pathway). In someembodiments, the oligonucleotides for use, or adaptable for use, targetNR1H3 mRNA and reduce the amount or level of NR1H3 mRNA, NR1H3 proteinand/or NR1H3 activity.

In addition, in some embodiments of the methods herein, a subject havinga disease, disorder, or condition associated with NR1H3 expression or ispredisposed to the same is selected for treatment with anoligonucleotide provided herein (e.g., an RNAi oligonucleotide). In someembodiments, the method comprises selecting an individual having amarker (e.g., a biomarker) for a disease, disorder, or conditionassociated with NR1H3 expression or predisposed to the same, such as,but not limited to, NR1H3 mRNA, NR1H3 protein, or a combination thereof.Likewise, and as detailed below, some embodiments of the methodsprovided by the disclosure include steps such as measuring or obtaininga baseline value for a marker of NR1H3 expression (e.g., NR1H3 mRNA),and then comparing such obtained value to one or more other baselinevalues or values obtained after the subject is administered theoligonucleotide to assess the effectiveness of treatment.

The disclosure also provides methods of treating a subject having,suspected of having, or at risk of developing a disease, disorder orcondition associated with a NR1H3 expression with an oligonucleotideprovided herein. In some respects, the current disclosure providesmethods of treating or attenuating the onset or progression of adisease, disorder or condition associated with NR1H3 expression usingthe oligonucleotides herein. In other aspects, the disclosure providesmethods to achieve one or more therapeutic benefits in a subject havinga disease, disorder, or condition associated with NR1H3 expression usingthe oligonucleotides provided herein. In some embodiments of the methodsherein, the subject is treated by administering a therapeuticallyeffective amount of any one or more of the oligonucleotides providedherein. In some embodiments, treatment comprises reducing NR1H3expression. In some embodiments, the subject is treated therapeutically.In some embodiments, the subject is treated prophylactically.

In some embodiments of the methods herein, one or more oligonucleotidesherein (e.g., RNAi oligonucleotides), or a pharmaceutical compositioncomprising one or more oligonucleotides, is administered to a subjecthaving a disease, disorder or condition associated with NR1H3 expressionsuch that NR1H3 expression is reduced in the subject, thereby treatingthe subject. In some embodiments, an amount or level of NR1H3 mRNA isreduced in the subject. In some embodiments, an amount or level of NR1H3protein is reduced in the subject. In some embodiments, an amount orlevel of NR1H3 activity is reduced in the subject.

In some embodiments of the methods herein, an oligonucleotide providedherein (e.g., an RNAi oligonucleotide), or a pharmaceutical compositioncomprising the oligonucleotide, is administered to a subject having adisease, disorder or condition associated with NR1H3 such that NR1H3expression is reduced in the subject by at least about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%,or greater than 99% when compared to NR1H3 expression prior toadministration of one or more oligonucleotides or pharmaceuticalcomposition. In some embodiments, NR1H3 expression is reduced in thesubject by at least about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 99%, or greater than 99% whencompared to NR1H3 expression in a subject (e.g., a reference or controlsubject) not receiving the oligonucleotide or oligonucleotides orpharmaceutical composition or receiving a control oligonucleotide oroligonucleotides, pharmaceutical composition or treatment.

In some embodiments of the methods herein, an oligonucleotide oroligonucleotides herein (e.g., RNAi oligonucleotides), or apharmaceutical composition comprising the oligonucleotide oroligonucleotides, is administered to a subject having a disease,disorder or condition associated with NR1H3 expression such that anamount or level of NR1H3 mRNA is reduced in the subject by at leastabout 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 99%, or greater than 99% when compared to the amount orlevel of NR1H3 mRNA prior to administration of the oligonucleotide orpharmaceutical composition. In some embodiments, an amount or level ofNR1H3 mRNA is reduced in the subject by at least about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%,or greater than 99% when compared to an amount or level of NR1H3 mRNA ina subject (e.g., a reference or control subject) not receiving theoligonucleotide or oligonucleotides or pharmaceutical composition orreceiving a control oligonucleotide or oligonucleotides, pharmaceuticalcomposition or treatment.

In some embodiments of the methods herein, an oligonucleotide oroligonucleotides herein, or a pharmaceutical composition comprising theoligonucleotide or oligonucleotides, is administered to a subject havinga disease, disorder or condition associated with NR1H3 expression suchthat an amount or level of NR1H3 protein is reduced in the subject by atleast about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99% or greater than 99% when compared to theamount or level of NR1H3 protein prior to administration of theoligonucleotide or pharmaceutical composition. In some embodiments, anamount or level of NR1H3 protein is reduced in the subject by at leastabout 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 99%, or greater than 99% when compared to an amount orlevel of NR1H3 protein in a subject (e.g., a reference or controlsubject) not receiving the oligonucleotide or oligonucleotides orpharmaceutical composition or receiving a control oligonucleotide,oligonucleotides or pharmaceutical composition or treatment.

In some embodiments of the methods herein, an oligonucleotide oroligonucleotides (e.g., RNAi oligonucleotides) herein, or apharmaceutical composition comprising the oligonucleotide oroligonucleotides, is administered to a subject having a disease,disorder or condition associated with NR1H3 such that an amount or levelof NR1H3 gene activity/expression is reduced in the subject by at leastabout 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 99%, or greater than 99% when compared to the amount orlevel of NR1H3 activity prior to administration of the oligonucleotideor pharmaceutical composition. In some embodiments, an amount or levelof NR1H3 activity is reduced in the subject by at least about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about99%, or greater than 99% when compared to an amount or level of NR1H3activity in a subject (e.g., a reference or control subject) notreceiving the oligonucleotide or pharmaceutical composition or receivinga control oligonucleotide, pharmaceutical composition or treatment.

Suitable methods for determining NR1H3 expression, the amount or levelof NR1H3 mRNA, NR1H3 protein, NR1H3 activity, or a biomarker related toor affected by modulation of NR1H3 expression (e.g., a plasmabiomarker), in the subject, or in a sample from the subject, are knownin the art. Further, the Examples set forth herein illustrate methodsfor determining NR1H3 expression.

In some embodiments, NR1H3 expression, the amount or level of NR1H3mRNA, NR1H3 protein, NR1H3 activity, or a biomarker related to oraffected by modulation of NR1H3 expression, or any combination thereof,is reduced in a cell (e.g., a hepatocyte), a population or a group ofcells (e.g., an organoid), an organ (e.g., liver), blood or a fractionthereof (e.g., plasma), a tissue (e.g., liver tissue), a sample (e.g., aliver biopsy sample), or any other appropriate biological materialobtained or isolated from the subject. In some embodiments, NR1H3expression, the amount or level of NR1H3 mRNA, NR1H3 protein, NR1H3activity, or a biomarker related to or affected by modulation of NR1H3expression, or any combination thereof, is reduced in more than one typeof cell (e.g., a hepatocyte and one or more other type(s) of cell), morethan one groups of cells, more than one organ (e.g., liver and one ormore other organ(s)), more than one fraction of blood (e.g., plasma andone or more other blood fraction(s)), more than one type of tissue(e.g., liver tissue and one or more other type(s) of tissue), or morethan one type of sample (e.g., a liver biopsy sample and one or moreother type(s) of biopsy sample).

Because of their high specificity, the oligonucleotides provided herein(e.g., dsRNAi oligonucleotides) specifically target mRNA of target genes(e.g., NR1H3 mRNA) of cells and tissue(s), or organs(s) (e.g., liver).In preventing disease, the target gene may be one which is required forinitiation or maintenance of the disease or which has been identified asbeing associated with a higher risk of contracting the disease. Intreating disease, the oligonucleotide can be brought into contact withthe cells, tissue(s), or organ(s) (e.g., liver) exhibiting orresponsible for mediating the disease. For example, an oligonucleotide(e.g., an RNAi oligonucleotide) substantially identical to all or partof a wild-type (i.e., native) or mutated gene associated with a disorderor condition associated with NR1H3 expression may be brought intocontact with or introduced into a cell or tissue type of interest suchas a hepatocyte or other liver cell.

In some embodiments, the target gene may be a target gene from anymammal, such as a human target. Any target gene may be silencedaccording to the method described herein.

Methods described herein typically involve administering to a subject aneffective amount of an oligonucleotide herein (e.g., a RNAioligonucleotide), that is, an amount that produces or generates adesirable therapeutic result. A therapeutically acceptable amount may bean amount that therapeutically treats a disease or disorder. Theappropriate dosage for any one subject will depend on certain factors,including the subject's size, body surface area, age, the composition tobe administered, the active ingredient(s) in the composition, time androute of administration, general health, and other drugs beingadministered concurrently.

In some embodiments, a subject is administered any one of thecompositions herein (e.g., a composition comprising an RNAioligonucleotide described herein) either enterally (e.g., orally, bygastric feeding tube, by duodenal feeding tube, via gastrostomy orrectally), parenterally (e.g., subcutaneous injection, intravenousinjection or infusion, intra-arterial injection or infusion,intraosseous infusion, intramuscular injection, intracerebral injection,intracerebroventricular injection, intrathecal), topically (e.g.,epicutaneous, inhalational, via eye drops, or through a mucousmembrane), or by direct injection into a target organ (e.g., the liverof a subject). Typically, oligonucleotides herein are administeredintravenously or subcutaneously.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide), or a pharmaceutical composition comprising theoligonucleotide, is administered alone or in combination. In someembodiments, the oligonucleotides herein are administered in combinationconcurrently, sequentially (in any order), or intermittently. Forexample, two oligonucleotides may be co-administered concurrently.Alternatively, one oligonucleotide may be administered and followed anyamount of time later (e.g., one hour, one day, one week or one month) bythe administration of a second oligonucleotide.

In some embodiments, an oligonucleotide herein (e.g., an RNAioligonucleotide), or a pharmaceutical composition comprising theoligonucleotide, is administered in combination with one or moreadditional pharmacologically active substances. In some embodiments theadditional pharmacologically active substances are selected from e.g.,anti-diabetic agents, anti-obesity agents, appetite regulating agents,antihypertensive agents, agents.

In some embodiments, the subject to be treated is a human or non-humanprimate or other mammalian subject. Other exemplary subjects includedomesticated animals such as dogs and cats; livestock such as horses,cattle, pigs, sheep, goats, and chickens; and animals such as mice,rats, guinea pigs, and hamsters.

Kits

In some embodiments, the disclosure provides a kit comprising anoligonucleotide herein (e.g., an RNAi oligonucleotide), and instructionsfor use. In some embodiments, the kit comprises an oligonucleotideherein, and a package insert containing instructions for use of the kitand/or any component thereof. In some embodiments, the kit comprises, ina suitable container, an oligonucleotide herein, one or more controls,and various buffers, reagents, enzymes and other standard ingredientswell known in the art. In some embodiments, the container comprises atleast one vial, well, test tube, flask, bottle, syringe, or othercontainer means, into which the oligonucleotide is placed, and in someinstances, suitably aliquoted. In some embodiments where an additionalcomponent is provided, the kit contains additional containers into whichthis component is placed. The kits can also include a means forcontaining the oligonucleotide and any other reagent in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained. Containers and/or kits can include labeling with instructionsfor use and/or warnings.

In some embodiments, a kit comprises an oligonucleotide herein (e.g., anRNAi oligonucleotide), and a pharmaceutically acceptable carrier, or apharmaceutical composition comprising the oligonucleotide andinstructions for treating or delaying progression of a disease, disorderor condition associated with NR1H3 expression in a subject in needthereof.

Definitions

As used herein, the term “antisense oligonucleotide” encompasses anucleic acid-based molecule which has a sequence complementary to all orpart of the target mRNA, in particular seed sequence thereby capable offorming a duplex with a mRNA. Thus, the term “antisenseoligonucleotide”, as used herein, may be referred to as “complementarynucleic acid-based inhibitor”.

As used herein, “approximately” or “about,” as applied to one or morevalues of interest, refers to a value that is similar to a statedreference value. In certain embodiments, “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

As used herein, “administer,” “administering,” “administration” and thelike refers to providing a substance (e.g., an oligonucleotide) to asubject in a manner that is pharmacologically useful (e.g., to treat adisease, disorder, or condition in the subject).

As used herein, “attenuate,” “attenuating,” “attenuation” and the likerefers to reducing or effectively halting. As a non-limiting example,one or more of the treatments herein may reduce or effectively halt theonset or progression of non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), or systemic lupus erythematosus ina subject. This attenuation may be exemplified by, for example, adecrease in one or more aspects (e.g., symptoms, tissue characteristics,and cellular, inflammatory, or immunological activity, etc.) ofnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), or systemic lupus erythematosus, no detectable progression(worsening) of one or more aspects fatty liver disease, or systemiclupus erythematosus, or no detectable aspects of non-alcoholic fattyliver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or systemiclupus erythematosus in a subject when they might otherwise be expected.

As used herein, “complementary” refers to a structural relationshipbetween two nucleotides (e.g., on two opposing nucleic acids or onopposing regions of a single nucleic acid strand) that permits the twonucleotides to form base pairs with one another. For example, a purinenucleotide of one nucleic acid that is complementary to a pyrimidinenucleotide of an opposing nucleic acid may base pair together by forminghydrogen bonds with one another. In some embodiments, complementarynucleotides can base pair in the Watson-Crick manner or in any othermanner that allows for the formation of stable duplexes. In someembodiments, two nucleic acids may have regions of multiple nucleotidesthat are complementary with each other to form regions ofcomplementarity, as described herein.

As used herein, “deoxyribonucleotide” refers to a nucleotide having ahydrogen in place of a hydroxyl at the 2′ position of its pentose sugarwhen compared with a ribonucleotide. A modified deoxyribonucleotide is adeoxyribonucleotide having one or more modifications or substitutions ofatoms other than at the 2′ position, including modifications orsubstitutions in or of the sugar, phosphate group or base.

As used herein, “double-stranded oligonucleotide” or “dsoligonucleotide” refers to an oligonucleotide that is substantially in aduplex form. In some embodiments, the complementary base-pairing ofduplex region(s) of a double-stranded oligonucleotide is formed betweenantiparallel sequences of nucleotides of covalently separate nucleicacid strands. In some embodiments, complementary base-pairing of duplexregion(s) of a double-stranded oligonucleotide is formed betweenantiparallel sequences of nucleotides of nucleic acid strands that arecovalently linked. In some embodiments, complementary base-pairing ofduplex region(s) of a double-stranded oligonucleotide is formed fromsingle nucleic acid strand that is folded (e.g., via a hairpin) toprovide complementary antiparallel sequences of nucleotides that basepair together. In some embodiments, a double-stranded oligonucleotidecomprises two covalently separate nucleic acid strands that are fullyduplexed with one another. However, in some embodiments, adouble-stranded oligonucleotide comprises two covalently separatenucleic acid strands that are partially duplexed (e.g., having overhangsat one or both ends). In some embodiments, a double-strandedoligonucleotide comprises antiparallel sequence of nucleotides that arepartially complementary, and thus, may have one or more mismatches,which may include internal mismatches or end mismatches.

As used herein, “duplex,” in reference to nucleic acids (e.g.,oligonucleotides), refers to a structure formed through complementarybase pairing of two antiparallel sequences of nucleotides.

As used herein, “excipient” refers to a non-therapeutic agent that maybe included in a composition, for example, to provide or contribute to adesired consistency or stabilizing effect.

As used herein, “hepatocyte” or “hepatocytes” refers to cells of theparenchymal tissues of the liver. These cells make up about 70-85% ofthe liver's mass and manufacture serum albumin, FBN and the prothrombingroup of clotting factors (except for Factors 3 and 4). Markers forhepatocyte lineage cells include, but are not limited to, transthyretin(Ttr), glutamine synthetase (Glu1), hepatocyte nuclear factor 1a (Hnf1a)and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytesmay include, but are not limited to, cytochrome P450 (Cyp3a11),fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin(Alb) and OC2-2F8. See, e.g., Huch et al. NATURE (2013); 494: 247-50.

As used herein, a “hepatotoxic agent” refers to a chemical compound,virus or other substance that is itself toxic to the liver or can beprocessed to form a metabolite that is toxic to the liver. Hepatotoxicagents may include, but are not limited to, carbon tetrachloride (CCl₄),acetaminophen (paracetamol), vinyl chloride, arsenic, chloroform,nonsteroidal anti-inflammatory drugs (such as aspirin andphenylbutazone).

As used herein, the term “NR1H3” refers to the gene which encodes to theprotein Liver X receptor alpha, or LXR-alpha. LXR-alpha is a nuclearreceptor protein that is encoded by NR1H3 (nuclear receptor subfamily 1,group H, member 3). The term “NR1H3” is intended to refer to allisoforms unless stated otherwise.

As used herein, “labile linker” refers to a linker that can be cleaved(e.g., by acidic pH). A “fairly stable linker” refers to a linker thatcannot be readily cleaved.

As used herein, “liver inflammation” or “hepatitis” refers to a physicalcondition in which the liver becomes swollen, dysfunctional and/orpainful, especially as a result of injury or infection, as may be causedby exposure to a hepatotoxic agent. Symptoms may include jaundice(yellowing of the skin or eyes), fatigue, weakness, nausea, vomiting,appetite reduction and weight loss. Liver inflammation, if leftuntreated, may progress to fibrosis, cirrhosis, liver failure or livercancer.

As used herein, “liver fibrosis” “Liver Fibrosis” or “fibrosis of theliver” refers to an excessive accumulation in the liver of extracellularmatrix proteins, which could include collagens (I, III, and IV), FBN,undulin, elastin, laminin, hyaluronan and proteoglycans resulting frominflammation and liver cell death. Liver fibrosis, if left untreated,may progress to cirrhosis, liver failure or liver cancer.

As used herein, “loop” refers to an unpaired region of a nucleic acid(e.g., oligonucleotide) that is flanked by two antiparallel regions ofthe nucleic acid that are sufficiently complementary to one another,such that under appropriate hybridization conditions (e.g., in aphosphate buffer, in a cell), the two antiparallel regions, which flankthe unpaired region, hybridize to form a duplex (referred to as a“stem”).

As used herein, “Metabolic syndrome’ or “metabolic liver disease” refersto a disorder characterized by a cluster of associated medicalconditions and associated pathologies including, but not limited to thefollowing medical conditions: abdominal obesity, elevated bloodpressure, elevated fasting plasma glucose, high serum triglycerides,liver fibrosis, and low levels of high-density lipoprotein (HDL) levels.As used herein, the term metabolic syndrome or metabolic liver diseasemay encompass a wide array of direct and indirect manifestations,diseases and pathologies associated with metabolic syndrome andmetabolic liver disease, with an expanded list of conditions usedthroughout the document.

As used herein, “modified internucleotide linkage” refers to aninternucleotide linkage having one or more chemical modifications whencompared with a reference internucleotide linkage comprising aphosphodiester bond. In some embodiments, a modified nucleotide is anon-naturally occurring linkage. Typically, a modified internucleotidelinkage confers one or more desirable properties to a nucleic acid inwhich the modified internucleotide linkage is present. For example, amodified internucleotide linkage may improve thermal stability,resistance to degradation, nuclease resistance, solubility,bioavailability, bioactivity, reduced immunogenicity, etc.

As used herein, “modified nucleotide” refers to a nucleotide having oneor more chemical modifications when compared with a correspondingreference nucleotide selected from: adenine ribonucleotide, guanineribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adeninedeoxyribonucleotide, guanine deoxyribonucleotide, cytosinedeoxyribonucleotide and thymidine deoxyribonucleotide. In someembodiments, a modified nucleotide is a non-naturally occurringnucleotide. In some embodiments, a modified nucleotide has one or morechemical modification in its sugar, nucleobase and/or phosphate group.In some embodiments, a modified nucleotide has one or more chemicalmoieties conjugated to a corresponding reference nucleotide. Typically,a modified nucleotide confers one or more desirable properties to anucleic acid in which the modified nucleotide is present. For example, amodified nucleotide may improve thermal stability, resistance todegradation, nuclease resistance, solubility, bioavailability,bioactivity, reduced immunogenicity, etc.

As used herein, “nicked tetraloop structure” refers to a structure of aRNAi oligonucleotide that is characterized by separate sense (passenger)and antisense (guide) strands, in which the sense strand has a region ofcomplementarity with the antisense strand, and in which at least one ofthe strands, generally the sense strand, has a tetraloop configured tostabilize an adjacent stem region formed within the at least one strand.

As used herein, “oligonucleotide” refers to a short nucleic acid (e.g.,less than about 100 nucleotides in length). An oligonucleotide may besingle-stranded (ss) or double-stranded (ds). An oligonucleotide may ormay not have duplex regions. As a set of non-limiting examples, anoligonucleotide may be, but is not limited to, a small interfering RNA(siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrateinterfering RNA (DsiRNA), antisense oligonucleotide, short siRNA or sssiRNA. In some embodiments, a double-stranded (dsRNA) is an RNAioligonucleotide.

As used herein, “overhang” refers to terminal non-base pairingnucleotide(s) resulting from one strand or region extending beyond theterminus of a complementary strand with which the one strand or regionforms a duplex. In some embodiments, an overhang comprises one or moreunpaired nucleotides extending from a duplex region at the 5′ terminusor 3′ terminus of an oligonucleotide. In certain embodiments, theoverhang is a 3′- or 5′-overhang on the antisense strand or sense strandof an oligonucleotide.

As used herein, “phosphate analog” refers to a chemical moiety thatmimics the electrostatic and/or steric properties of a phosphate group.In some embodiments, a phosphate analog is positioned at the 5′ terminalnucleotide of an oligonucleotide in place of a 5′-phosphate, which isoften susceptible to enzymatic removal. In some embodiments, a 5′phosphate analog contains a phosphatase-resistant linkage. Examples ofphosphate analogs include, but are not limited to, 5′ phosphonates, suchas 5′ methylene phosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP).In some embodiments, an oligonucleotide has a phosphate analog at a4′-carbon position of the sugar (referred to as a “4′-phosphate analog”)at a 5′-terminal nucleotide. An example of a 4′-phosphate analog isoxymethylphosphonate, in which the oxygen atom of the oxymethyl group isbound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof.See, e.g., US Provisional Patent Application Nos. 62/383,207 (filed on 2Sep. 2016) and 62/393,401 (filed on 12 Sep. 2016). Other modificationshave been developed for the 5′ end of oligonucleotides (see, e.g., Intl.Patent Application No. WO 2011/133871; U.S. Pat. No. 8,927,513; andPrakash et al. NUCLEIC ACIDS RES. (2015); 43: 2993-3011).

As used herein, “reduced expression” of a gene (e.g., NR1H3) refers to adecrease in the amount or level of RNA transcript (e.g., NR1H3 mRNA) orprotein encoded by the gene and/or a decrease in the amount or level ofactivity of the gene in a cell, a population of cells, a sample, or asubject, when compared to an appropriate reference (e.g., a referencecell, population of cells, sample or subject). For example, the act ofcontacting a cell with an oligonucleotide herein (e.g., anoligonucleotide comprising an antisense strand having a nucleotidesequence that is complementary to a nucleotide sequence comprising NR1H3mRNA) may result in a decrease in the amount or level of NR1H3 mRNA,protein and/or activity (e.g., via degradation of NR1H3 mRNA by the RNAipathway) when compared to a cell that is not treated with theoligonucleotide. Similarly, and as used herein, “reducing expression”refers to an act that results in reduced expression of a gene (e.g.,NR1H3).

As used herein, “reduction of NR1H3 expression” refers to a decrease inthe amount or level of NR1H3 mRNA, NR1H3 protein and/or NR1H3 activityin a cell, a population of cells, a sample or a subject when compared toan appropriate reference (e.g., a reference cell, population of cells,sample, or subject).

As used herein, “region of complementarity” refers to a sequence ofnucleotides of a nucleic acid (e.g., an oligonucleotide) that issufficiently complementary to an antiparallel sequence of nucleotides topermit hybridization between the two sequences of nucleotides underappropriate hybridization conditions (e.g., in a phosphate buffer, in acell, etc.). In some embodiments, an oligonucleotide herein comprises atargeting sequence having a region of complementary to a mRNA targetsequence.

As used herein, “ribonucleotide” refers to a nucleotide having a riboseas its pentose sugar, which contains a hydroxyl group at its 2′position. A modified ribonucleotide is a ribonucleotide having one ormore modifications or substitutions of atoms other than at the 2′position, including modifications or substitutions in or of the ribose,phosphate group or base.

As used herein, “RNAi oligonucleotide” refers to either (a) adouble-stranded oligonucleotide having a sense strand (passenger) andantisense strand (guide), in which the antisense strand or part of theantisense strand is used by the Argonaute 2 (Ago2) endonuclease in thecleavage of a target mRNA (e.g., NR1H3 mRNA) or (b) a single-strandedoligonucleotide having a single antisense strand, where that antisensestrand (or part of that antisense strand) is used by the Ago2endonuclease in the cleavage of a target mRNA (e.g., NR1H3 mRNA).

As used herein, “strand” refers to a single, contiguous sequence ofnucleotides linked together through internucleotide linkages (e.g.,phosphodiester linkages or phosphorothioate linkages). In someembodiments, a strand has two free ends (e.g., a 5′ end and a 3′ end).

As used herein, “subject” means any mammal, including mice, rabbits, andhumans. In one embodiment, the subject is a human or NHP. Moreover,“individual” or “patient” may be used interchangeably with “subject.”

As used herein, “synthetic” refers to a nucleic acid or other moleculethat is artificially synthesized (e.g., using a machine (e.g., asolid-state nucleic acid synthesizer)) or that is otherwise not derivedfrom a natural source (e.g., a cell or organism) that normally producesthe molecule.

As used herein, “targeting ligand” refers to a molecule (e.g., acarbohydrate, amino sugar, cholesterol, polypeptide, or lipid) thatselectively binds to a cognate molecule (e.g., a receptor) of a tissueor cell of interest and that is conjugatable to another substance forpurposes of targeting the other substance to the tissue or cell ofinterest. For example, in some embodiments, a targeting ligand may beconjugated to an oligonucleotide for purposes of targeting theoligonucleotide to a specific tissue or cell of interest. In someembodiments, a targeting ligand selectively binds to a cell surfacereceptor. Accordingly, in some embodiments, a targeting ligand whenconjugated to an oligonucleotide facilitates delivery of theoligonucleotide into a particular cell through selective binding to areceptor expressed on the surface of the cell and endosomalinternalization by the cell of the complex comprising theoligonucleotide, targeting ligand and receptor. In some embodiments, atargeting ligand is conjugated to an oligonucleotide via a linker thatis cleaved following or during cellular internalization such that theoligonucleotide is released from the targeting ligand in the cell. Insome embodiments, the targeting ligand comprises at least one GalNAcmoiety and targets the liver and human liver cells (e.g., humanhepatocytes).

As used herein, “tetraloop” refers to a loop that increases stability ofan adjacent duplex formed by hybridization of flanking sequences ofnucleotides. The increase in stability is detectable as an increase inmelting temperature (T_(m)) of an adjacent stem duplex that is higherthan the T_(m) of the adjacent stem duplex expected, on average, from aset of loops of comparable length consisting of randomly selectedsequences of nucleotides. For example, a tetraloop can confer a T_(m) ofat least about 50° C., at least about 55° C., at least about 56° C., atleast about 58° C., at least about 60° C., at least about 65° C., or atleast about 75° C. in 10 mM Na₂HPO₄ to a hairpin comprising a duplex ofat least 2 base pairs (bp) in length. In some embodiments, a tetraloopcan confer a Tm of at least about 50° C., at least about 55° C., atleast about 56° C., at least about 58° C., at least about 60° C., atleast about 65° C., or at least about 75° C. in 10 mM NaH₂PO4 to ahairpin comprising a duplex of at least 2 base pairs (bp) in length. Insome embodiments, a tetraloop may stabilize a bp in an adjacent stemduplex by stacking interactions. In addition, interactions among thenucleotides in a tetraloop include, but are not limited to,non-Watson-Crick base pairing, stacking interactions, hydrogen bondingand contact interactions (Cheong et al. NATURE (1990); 346: 680-82; andHeus & Pardi SCIENCE (1991); 253: 191-94). In some embodiments, atetraloop comprises or consists of 3 to 6 nucleotides and is typically 4to 5 nucleotides. In certain embodiments, a tetraloop comprises orconsists of 3, 4, 5, or 6 nucleotides, which may or may not be modified(e.g., which may or may not be conjugated to a targeting moiety). In oneembodiment, a tetraloop consists of 4 nucleotides. Any nucleotide may beused in the tetraloop and standard IUPAC-IUB symbols for suchnucleotides may be used as described in Cornish-Bowden NUCLEIC ACIDSRES. (1985); 13: 3021-30. For example, the letter “N” may be used tomean that any base may be in that position, the letter “R” may be usedto show that A (adenine) or G (guanine) may be in that position, and “B”may be used to show that C (cytosine), G (guanine), or T (thymine) maybe in that position. Examples of tetraloops include the UNCG family oftetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), andthe CUUG tetraloop (Woese et al. PROC. NATL. ACAD. SCI. USA (1990); 87:8467-71; Antao et al. NUCLEIC ACIDS RES. (1991); 19: 5901-05). Examplesof DNA tetraloops include the d(GNNA) family of tetraloops (e.g.,d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family oftetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family oftetraloops (e.g., d(TTCG)). See, e.g., Nakano et al. BIOCHEM. (2002);41: 14281-92; Shinji et al. NIPPON KAGAKKAI KOEN YOKOSHU (2000); 78:731. In some embodiments, the tetraloop is contained within a nickedtetraloop structure.

As used herein, “treat” or “treating” refers to the act of providingcare to a subject in need thereof, for example, by administering atherapeutic agent (e.g., an oligonucleotide herein) to the subject, forpurposes of improving the health and/or well-being of the subject withrespect to an existing condition (e.g., a disease, disorder) or toprevent or decrease the likelihood of the occurrence of a condition. Insome embodiments, treatment involves reducing the frequency or severityof at least one sign, symptom or contributing factor of a condition(e.g., disease, disorder) experienced by a subject.

EXAMPLES

While the disclosure has been described with reference to the specificembodiments set forth in the following Examples, it should be understoodby those skilled in the art that various changes may be made, andequivalents may be substituted without departing from the true spiritand scope of the disclosure. Further, the following Examples are offeredby way of illustration and are not intended to limit the scope of thedisclosure in any manner. In addition, modifications may be made toadapt to a situation, material, composition of matter, process, processstep or steps, to the objective, spirit, and scope of the disclosure.All such modifications are intended to be within the scope of thedisclosure. Standard techniques well known in the art or the techniquesspecifically described below were utilized.

Example 1: Preparation of RNAi Oligonucleotides OligonucleotideSynthesis and Purification

The oligonucleotides (RNAi oligonucleotides) described in the foregoingExamples are chemically synthesized using methods described herein.Generally, RNAi oligonucleotides are synthesized using solid phaseoligonucleotide synthesis methods as described for 19-23mer siRNAs (see,e.g., Scaringe et al. NUCLEIC ACIDS RES. (1990); 18: 5433-41 and Usmanet al. J. AM. CHEM. SOC. (1987); 109: 7845-45; see also, U.S. Pat. Nos.5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657;6,353,098; 6,362,323; 6,437,117 and 6,469,158) in addition to usingknown phosphoramidite synthesis (see, e.g. Hughes and Ellington, COLDSPRING HARB. PERSPECT. BIOL. (2017); 9(1): a023812; Beaucage S. L.,Caruthers M. H. Studies on Nucleotide Chemistry V. DeoxynucleosidePhosphoramidites—A New Class of Key Intermediates forDeoxypolynucleotide Synthesis, TETRAHEDRON LETT. (1981); 22: 1859-62.doi:10.1016/S0040-4039(01)90461-7). dsRNAi oligonucleotides having a19mer core sequence were formatted into constructs having a 25mer sensestrand and a 27mer antisense strand to allow for processing by the RNAimachinery. The 19mer core sequence is complementary to a region in theNR1H3 mRNA.

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies; Coralville, Iowa). Forexample, RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech; Piscataway, N.J.) using standard techniques(Damha & Olgivie, METHODS MOL. BIOL. (1993); 20: 81-114; Wincott et al.NUCLEIC ACIDS RES. (1995); 23: 2677-84). The oligomers were purifiedusing ion-exchange high performance liquid chromatography (IE-HPLC) onan Amersham Source 15Q column (1.0 cm×25 cm; Amersham Pharmacia Biotech)using a 15 min step-linear gradient. The gradient varied from 90:10Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at260 nm and peaks corresponding to the full-length oligonucleotidespecies were collected, pooled, desalted on NAP-5 columns, andlyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, Calif.).The CE capillaries have a 100 m inner diameter and contain ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, run in an electric field of 444 V/cm, and wasdetected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere obtained that were at least 90% pure as assessed by CE for use inexperiments described below. Compound identity was verified bymatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectroscopy on a Voyager DE™ Biospectometry Work Station (AppliedBiosystems; Foster City, Calif.) following the manufacturer'srecommended protocol. Relative molecular masses of all oligomers wereobtained, often within 0.2% of expected molecular mass.

Preparation of Duplexes

Single strand RNA oligomers were resuspended (e.g., at 100 μMconcentration) in duplex buffer consisting of 100 mM potassium acetate,30 mM HEPES, pH 7.5. Complementary sense and antisense strands weremixed in equal molar amounts to yield a final solution of, for example,50 μM duplex. Samples were heated to 100° C. for 5′ in RNA buffer (IDT)and were allowed to cool to room temperature before use. The RNAioligonucleotides were stored at −20° C. Single strand RNA oligomers werestored lyophilized or in nuclease-free water at −80° C.

Example 2: Generation of NR1H3-Targeting Double Stranded RNAiOligonucleotides

Identification of NR1H3 mRNA Target Sequences

Nuclear Receptor Subfamily 1 Group H Member 3 (NR1H3) is a protein thatregulates macrophage function, lipid homeostasis, and inflammation. Togenerate RNAi oligonucleotide inhibitors of NR1H3 expression, acomputer-based algorithm was used to computationally identify NR1H3 mRNAtarget sequences suitable for assaying inhibition of NR1H3 expression bythe RNAi pathway. The algorithm provided RNAi oligonucleotide guide(antisense) strand sequences each having a region of complementarity toa suitable NR1H3 target sequence of human NR1H3 mRNA. Some of the guidestrand sequences identified by the algorithm were also complementary tothe corresponding NR1H3 target sequence of monkey and/or mouse NR1H3mRNA. NR1H3 RNAi oligonucleotides comprising a region of complementarityto homologous NR1H3 mRNA target sequences with nucleotide sequencesimilarity are predicted to have the ability to target homologous NR1H3mRNAs.

RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) weregenerated as described in Example 1 for evaluation in vitro. Each DsiRNAwas generated with the same modification pattern, and each with a uniqueguide strand having a region of complementarity to a NR1H3 targetsequence identified by the algorithm. Modifications for the sense andanti-sense DsiRNA included the following (X—any nucleotide;m—2′-O-methyl modified nucleotide; r—ribosyl modified nucleotide):

Sense Strand: rXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXXXAnti-sense Strand: mXmXmXmXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXmXmX

In Vitro Cell-Based Assays

The ability of each of the modified DsiRNA in Table 1 to reduce NR1H3mRNA was measured using in vitro cell-based assays. Briefly, human Huh-7cells expressing endogenous human NR1H3 gene were transfected with eachof the DsiRNAs listed in Table 1 at 1 nM in separate wells of amulti-well cell-culture plate. Cells were maintained for 24 hoursfollowing transfection with the modified DsiRNA, and then the amount ofremaining NR1H3 mRNA from the transfected cells was determined using andRT-qPCR assay. The assay used the following primers and probe normalizedto the geometric mean of two reference genes HPRT1 and SFRS9:Forward-1198 GTTATAACCGGGAAGACTTTGC (SEQ ID NO:1122); Reverse-1326:TGATAGCAATGAGCAAGGCA (SEQ ID NO: 1123); Probe-1253:ATGGCCCTGGAGAACTCGAAGATG (SEQ ID NO:1124). The primer pair was assayedfor % remaining RNA as shown in Table 1. DsiRNAs resulting in less thanor equal to 10% NR1H3 mRNA remaining in DsiRNA-transfected cells whencompared to mock-transfected cells were considered DsiRNA “hits”. TheHuH-7 cell-based assay evaluating the ability of the DsiRNAs listed inTable 1 to inhibit NR1H3 expression identified several candidateDsiRNAs.

Taken together, these results show that DsiRNAs designed to target humanNR1H3 mRNA inhibit NR1H3 expression in cells, as determined by a reducedamount of NR1H3 mRNA in DsiRNA-transfected cells relative to controlcells. These results demonstrate that the nucleotide sequencescomprising the DsiRNA are useful for generating RNAi oligonucleotides toinhibit NR1H3 expression. Further, these results demonstrate thatmultiple NR1H3 mRNA target sequences are suitable for the RNAi-mediatedinhibition of NR1H3 expression.

TABLE 1 In Vitro Screening Results SED ID NO SED ID NO (Sense(Anti-sense NR1H3-F1198 Strand) Strand) DsiRNA name % remaining SEM 1385 NR1H3-764-784-861 8.9 4.43 2 386 NR1H3-766-786-863 16.25 3.11 3 387NR1H3-789-809-886 15.08 1.03 4 388 NR1H3-790-810-887 18.74 3.03 5 389NR1H3-791-811-888 40.34 2.62 6 390 NR1H3-792-812-889 32.44 4.47 7 391NR1H3-793-813-890 33.77 2.89 8 392 NR1H3-795-815-892 10.87 4.86 9 393NR1H3-796-816-893 14.94 1.85 10 394 NR1H3-797-817-894 45.07 9.79 11 395NR1H3-798-818-895 17.91 4.71 12 396 NR1H3-799-819-896 17.87 2.13 13 397NR1H3-802-822-899 8.47 3.71 14 398 NR1H3-803-823-900 26.43 3.01 15 399NR1H3-804-824-901 13.60 2.34 16 400 NR1H3-806-826-903 47.05 7.86 17 401NR1H3-808-828-905 14.07 1.86 18 402 NR1H3-809-829-906 37.06 10.23 19 403NR1H3-810-830-907 27.90 12.22 20 404 NR1H3-811-831-908 41.69 8.58 21 405NR1H3-813-833-910 29.81 2.83 22 406 NR1H3-844 22.31 3.43 23 407NR1H3-895-915-992 13.4 2.84 24 408 NR1H3-898-918-995 21.63 3.63 25 409NR1H3-915-935 23.64 3.59 26 410 NR1H3-917-937 13.58 3.29 27 411NR1H3-922-942 21.35 3.75 28 412 NR1H3-924-944 32.89 16.44 29 413NR1H3-925-945 15.74 4.69 30 414 NR1H3-927-947 26.60 5.18 31 415NR1H3-928-948 14.55 3.21 32 416 NR1H3-929-949 9.89 1.40 33 417NR1H3-930-950 26.20 3.86 34 418 NR1H3-931-951 27.44 1.62 35 419NR1H3-932-952 12.31 6.33 36 420 NR1H3-933-953 13.29 1.84 37 421NR1H3-941-961 10.08 2.10 38 422 NR1H3-944-964 13.78 1.05 39 423NR1H3-945-965 27.16 2.75 40 424 NR1H3-946-966 15.99 1.88 41 425NR1H3-947-967 8.63 3.01 42 426 NR1H3-949-969 17.43 1.64 43 427NR1H3-951-971 9.66 1.70 44 428 NR1H3-952-972 9.92 1.39 45 429NR1H3-953-973 21.75 4.12 46 430 NR1H3-1151-1171 18.83 2.78 47 431NR1H3-1153-1173 78.76 9.71 48 432 NR1H3-1154-1174 45.38 5.78 49 433NR1H3-1155-1175 23.72 2.37 50 434 NR1H3-1156-1176 23.75 2.37 51 435NR1H3-1157-1177 67.42 6.83 52 436 NR1H3-1158-1178 17.98 2.57 53 437NR1H3-1159-1179 17.47 2.49 54 438 NR1H3-1159-1180 9.14 1.61 55 439NR1H3-1161-1181 22.52 2.41 56 440 NR1H3-1162-1182 10.89 1.66 57 441NR1H3-1163-1183 38.50 7.62 58 442 NR1H3-1164-1184 23.52 9.14 59 443NR1H3-1165-1185 22.81 3.68 60 444 NR1H3-1166-1186 23.01 4.64 61 445NR1H3-1167-1187 50.54 10.76 62 446 NR1H3-1169-1189 15.60 2.02 63 447NR1H3-1170-1190 22.78 5.79 64 448 NR1H3-1171-1191 80.98 9.76 65 449NR1H3-1173-1193 16.14 4.16 66 450 NR1H3-1175-1195 45.81 7.47 67 451NR1H3-1176-1196 18.32 4.59 68 452 NR1H3-1177-1197 26.85 5.22 69 453NR1H3-1178-1198 63.10 17.52 70 454 NR1H3-1179-1199 12.86 1.65 71 455NR1H3-1180-1200 20.97 4.62 72 456 NR1H3-1181-1201 16.21 4.23 73 457NR1H3-1182-1202 30.61 5.64 74 458 NR1H3-1183-1203 30.96 6.72 75 459NR1H3-1184-1204 23.71 6.05 76 460 NR1H3-1185-1205 14.77 1.30 77 461NR1H3-1186-1206 19.94 6.28 78 462 NR1H3-1187-1207 14.48 1.77 79 463NR1H3-1188-1208 37.28 3.76 80 464 NR1H3-1190-1210 38.58 4.63 81 465NR1H3-1191-1211 15.02 2.19 82 466 NR1H3-1192-1212 51.23 7.43 83 467NR1H3-1193-1213 17.05 3.23 84 468 NR1H3-1194-1214 31.91 6.24 85 469NR1H3-1196-1216 32.00 9.03 86 470 NR1H3-1197-1217 8.36 2.55 87 471NR1H3-1198-1218 29.10 4.48 88 472 NR1H3-1199-1219 13.22 2.89 89 473NR1H3-1200-1220 8.59 2.38 90 474 NR1H3-1203-1223 15.92 3.29 91 475NR1H3-1204-1224 5.07 0.83 92 476 NR1H3-1207-1227 3.54 2.18 93 477NR1H3-1211-1231 1.88 1.19 94 478 NR1H3-1212-1232 17.48 7.55 95 479NR1H3-1213-1233 8.32 4.17 96 480 NR1H3-1214-1234 16.53 1.70 97 481NR1H3-1215-1235 11.84 1.93 98 482 NR1H3-1216-1236 9.48 1.67 99 483NR1H3-1217-1237 11.9 2.34 100 484 NR1H3-1218-1238 8.45 1.98 101 485NR1H3-1219-1239 6.80 1.35 102 486 NR1H3-1220-1240 3.60 1.88 103 487NR1H3-1222-1242 8.53 1.67 104 488 NR1H3-1223-1243 12.69 1.63 105 489NR1H3-1224-1244 6.15 2.42 106 490 NR1H3-1225-1245 9.72 3.87 107 491NR1H3-1226-1246 47.24 5.54 108 492 NR1H3-1227-1247 13.80 2.63 109 493NR1H3-1228-1248 6.76 1.78 110 494 NR1H3-1229-1249 8.75 2.71 111 495NR1H3-1232-1252 36.21 3.20 112 496 NR1H3-1233-1253 20.37 2.75 113 497NR1H3-1234-1254 18.42 1.50 114 498 NR1H3-1235-1255 16.37 1.48 115 499NR1H3-1236-1256 58.83 6.78 116 500 NR1H3-1237-1257 16.03 2.29 117 501NR1H3-1238-1258 19.29 5.59 118 502 NR1H3-1241-1261 12.44 2.25 119 503NR1H3-1242-1262 29.53 10.05 120 504 NR1H3-1243-1263 6.22 5.33 121 505NR1H3-1244-1264 5.51 0.94 122 506 NR1H3-1245-1265 15.00 2.82 123 507NR1H3-1246-1266 10.14 2.84 124 508 NR1H3-1247-1267 13.70 3.55 125 509NR1H3-1248-1268 37.81 4.12 126 510 NR1H3-1250-1270 10.08 2.10 127 511NR1H3-1251-1271 6.50 0.47 128 512 NR1H3-1252-1272 3.35 0.65 129 513NR1H3-1253-1273 14.22 1.35 130 514 NR1H3-1256-1276 8.26 1.63 131 515NR1H3-1258-1278 7.38 1.26 132 516 NR1H3-1259-1279 4.72 1.13 133 517NR1H3-1261-1281 4.39 1.31 134 518 NR1H3-1262-1282 27.66 5.46 135 519NR1H3-1265-1285 3.35 1.10 136 520 NR1H3-1266-1286 4.91 0.83 137 521NR1H3-1267-1287 21.91 4.48 138 522 NR1H3-1268-1288 21.59 3.96 139 523NR1H3-1269-1289 11.31 1.44 140 524 NR1H3-1270-1290 9.87 5.24 141 525NR1H3-1271-1291 6.95 1.9 142 526 NR1H3-1272-1292 7.07 0.99 143 527NR1H3-1273-1293 11.01 3.72 144 528 NR1H3-1275-1295 18.49 2.31 145 529NR1H3-1276-1296 17.77 3.25 146 530 NR1H3-1277-1297 18.20 1.62 147 531NR1H3-1278-1298 9.35 1.70 148 532 NR1H3-1279-1299 8.96 1.60 149 533NR1H3-1280-1300 19.16 3.95 150 534 NR1H3-1281-1301 12.33 1.94 151 535NR1H3-1282-1302 40.52 7.51 152 536 NR1H3-1283-1303 15.26 7.23 153 537NR1H3-1284-1304 37.09 9.79 154 538 NR1H3-1285-1305 29.28 4.88 155 539NR1H3-1286-1306 41.56 4.92 156 540 NR1H3-1288-1308 68.84 4.13 157 541NR1H3-1289-1309 34.44 10.86 158 542 NR1H3-1290-1310 17.67 3.83 159 543NR1H3-1291-1311 21.47 2.26 160 544 NR1H3-1292-1312 41.99 7.72 161 545NR1H3-1293-1313 13.77 1.40 162 546 NR1H3-1294-1314 20.97 3.76 163 547NR1H3-1295-1315 14.39 3.21 164 548 NR1H3-1296-1316 28.04 10.25 165 549NR1H3-1297-1317 12.70 2.20 166 550 NR1H3-1338-1358 22.20 4.81 167 551NR1H3-1339-1359 7.86 1.5 168 552 NR1H3-1340-1360 4.72 1.10 169 553NR1H3-1341-1361 11.49 4.26 170 554 NR1H3-1342-1362 4.84 1.18 171 555NR1H3-1343-1363 17.05 4.21 172 556 NR1H3-1344-1364 13.83 6.27 173 557NR1H3-1345-1365 9.55 1.22 174 558 NR1H3-1346-1366 4.54 1.14 175 559NR1H3-1347-1367 6.47 1.87 176 560 NR1H3-1377-1443 46.13 5.08 177 561NR1H3-1379-1445 28.21 2.69 178 562 NR1H3-1383-1449 27.33 3.23 179 563NR1H3-1384-1450 27.81 4.58 180 564 NR1H3-1385-1451 44.36 4.37 181 565NR1H3-1387-1453 15.50 2.5 182 566 NR1H3-1388-1454 46.03 6.42 183 567NR1H3-1391-1457 20.33 2.82 184 568 NR1H3-1393-1459 27.25 2.95 185 569NR1H3-1394-1460 11.31 2.71 186 570 NR1H3-1395-1461 30.82 3.24 187 571NR1H3-1396-1462 34.98 4.45 188 572 NR1H3-1397-1463 41.12 4.28 189 573NR1H3-1398-1464 14.14 2.79 190 574 NR1H3-1399-1465 14.46 2.61 191 575NR1H3-1400-1466 49.48 5.89 192 576 NR1H3-1401-1467 21.19 3.39 193 577NR1H3-1402-1468 14.17 2.73 194 578 NR1H3-1403-1469 18.25 1.29 195 579NR1H3-1404-1470 34.21 2.29 196 580 NR1H3-1406-1472 46.79 7.22 197 581NR1H3-1407-1473 13.59 2.47 198 582 NR1H3-1408-1474 44.63 5.37 199 583NR1H3-1410-1476 43.93 9.37 200 584 NR1H3-1411-1477 28.12 4.11 201 585NR1H3-1412-1478 50.22 11.35 202 586 NR1H3-1413-1479 45.56 6.23 203 587NR1H3-1414-1480 56.42 9.61 204 588 NR1H3-1415-1481 32.43 10.32 205 589NR1H3-1416-1482 16.66 2.27 206 590 NR1H3-1417-1483 31.84 4.71 207 591NR1H3-1418-1484 7.07 0.80 208 592 NR1H3-1419-1485 69.83 8.81 209 593NR1H3-1420-1486 16.01 5.34 210 594 NR1H3-1421-1487 38.29 6.31 211 595NR1H3-1422-1488 80.48 13.59 212 596 NR1H3-1423-1489 9.79 1.47 213 597NR1H3-1424-1490 9.36 1.84 214 598 NR1H3-1425-1491 22.14 3.77 215 599NR1H3-1426-1492 9.86 2.14 216 600 NR1H3-1427-1493 18.88 6.80 217 601NR1H3-1428-1494 17.55 5.52 218 602 NR1H3-1429-1495 2.77 1.08 219 603NR1H3-1430-1496 19.29 3.14 220 604 NR1H3-1431-1497 8.66 3.71 221 605NR1H3-1432-1498 19.76 3.07 222 606 NR1H3-1433-1499 3.37 2.29 223 607NR1H3-1434-1500 77.93 11.83 224 608 NR1H3-1435-1501 14.53 5.77 225 609NR1H3-1436-1502 11.42 2.45 226 610 NR1H3-1437-1503 4.97 1.57 227 611NR1H3-1438-1504 7.40 1.89 228 612 NR1H3-1439-1505 3.26 0.97 229 613NR1H3-1440-1506 40.21 5.31 230 614 NR1H3-1442-1508 50.09 3.47 231 615NR1H3-1443-1509 11.50 5.54 232 616 NR1H3-1444-1510 29.36 15.66 233 617NR1H3-1445-1511 47.59 6.76 234 618 NR1H3-1446-1512 2.37 0.65 235 619NR1H3-1447-1513 10.29 4.95 236 620 NR1H3-1448-1514 45.74 4.52 237 621NR1H3-1449-1515 35.27 4.39 238 622 NR1H3-1450-1516 39.10 5.63 239 623NR1H3-1451-1517 9.58 2.83 240 624 NR1H3-1452-1518 64.45 15.14 241 625NR1H3-1453-1519 14.78 2.76 242 626 NR1H3-1454-1520 5.01 2.14 243 627NR1H3-1455-1521 21.75 3.59 244 628 NR1H3-1456-1522 9.11 2.01 245 629NR1H3-1457-1523 7.52 3.29 246 630 NR1H3-1459-1525 7.81 1.75 247 631NR1H3-1460-1526 4.57 1.07 248 632 NR1H3-1461-1527 30.77 4.42 249 633NR1H3-1462-1528 6.29 1.92 250 634 NR1H3-1463-1529 2.58 0.85 251 635NR1H3-1465-1531 4.17 1.14 252 636 NR1H3-1466-1532 15.04 1.90 253 637NR1H3-1468-1534 14.47 1.37 254 638 NR1H3-1469-1535 14.04 2.26 255 639NR1H3-1471-1537 14.94 1.55 256 640 NR1H3-1472-1538 45.97 3.01 257 641NR1H3-1473-1539 19.37 8.93 258 642 NR1H3-1474-1540 9.53 2.70 259 643NR1H3-1475-1541 3.21 1.94 260 644 NR1H3-1476-1542 11.27 4.12 261 645NR1H3-1477-1543 14.30 2.78 262 646 NR1H3-1478-1544 13.06 3.66 263 647NR1H3-1479-1545 3.97 0.86 264 648 NR1H3-1480-1546 4.82 1.41 265 649NR1H3-1481-1547 2.81 0.57 266 650 NR1H3-1483-1549 5.56 1.60 267 651NR1H3-1484-1550 20.18 2.39 268 652 NR1H3-1485-1551 3.52 2.10 269 653NR1H3-1486-1552 13.41 1.81 270 654 NR1H3-1487-1553 25.35 3.42 271 655NR1H3-1488-1554 28.45 6.63 272 656 NR1H3-1489-1555 15.31 5.00 273 657NR1H3-1491-1557 6.33 1.03 274 658 NR1H3-1492-1558 20.75 4.42 275 659NR1H3-1494-1560 24.97 4.76 276 660 NR1H3-1505-1571 38.74 5.88 277 661NR1H3-1507-1573 9.42 2.29 278 662 NR1H3-1508-1574 36.57 6.54 279 663NR1H3-1509-1575 29.82 5.45 280 664 NR1H3-1510-1576 19.45 3.79 281 665NR1H3-1511-1577 23.73 3.3 282 666 NR1H3-1512-1578 42.41 7.68 283 667NR1H3-1513-1579 33.80 5.58 284 668 NR1H3-1514-1580 26.85 4.79 285 669NR1H3-1515-1581 3.95 0.84 286 670 NR1H3-1516-1582 16.48 3.36 287 671NR1H3-1517-1583 4.38 1.57 288 672 NR1H3-1518-1584 3.43 1.32 289 673NR1H3-1519-1585 5.73 1.72 290 674 NR1H3-1520-1586 14.37 1.95 291 675NR1H3-1521-1587 15.60 9.44 292 676 NR1H3-1522-1588 14.40 3.9 293 677NR1H3-1523-1589 9.60 2.11 294 678 NR1H3-1525-1591 13.51 4.15 295 679NR1H3-1526-1592 50.59 16.74 296 680 NR1H3-1527-1593 8.47 4.27 297 681NR1H3-1528-1594 25.44 3.59 298 682 NR1H3-1529-1595 12.55 2.77 299 683NR1H3-1530-1596 11.6 3.15 300 684 NR1H3-1531-1597 6.14 2.83 301 685NR1H3-1532-1598 15.35 4.55 302 686 NR1H3-1533-1599 3.13 3 303 687NR1H3-1534-1600 8.51 3.07 304 688 NR1H3-1535-1601 1.48 0.67 305 689NR1H3-1536-1602 30.02 10.69 306 690 NR1H3-1537-1603 17.55 6.31 307 691NR1H3-1538-1604 21.90 1.81 308 692 NR1H3-1539-1605 28.62 6.45 309 693NR1H3-1540-1606 19.29 5.08 310 694 NR1H3-1541-1607 10.51 2.94 311 695NR1H3-1542-1608 8.39 6.98 312 696 NR1H3-1543-1609 5.68 3.86 313 697NR1H3-1544-1610 23.78 4.02 314 698 NR1H3-1545-1611 3.93 1.19 315 699NR1H3-1546-1612 11.55 1.89 316 700 NR1H3-1547-1613 16.77 3.13 317 701NR1H3-1548-1614 10.86 7.45 318 702 NR1H3-1549-1615 33.40 8.27 319 703NR1H3-1550-1616 13.98 3.23 320 704 NR1H3-1551-1617 32.11 9.43 321 705NR1H3-1553-1619 43.83 11.06 322 706 NR1H3-1554-1620 5.16 1.29 323 707NR1H3-1555-1621 89.05 22.10 324 708 NR1H3-1556-1622 33.15 4.79 325 709NR1H3-1558-1624 15.27 4.11 326 710 NR1H3-1559-1625 15.15 3.47 327 711NR1H3-1560-1626 34.65 4.83 328 712 NR1H3-1561-1627 63.67 7.04 329 713NR1H3-1562-1628 16.78 10.54 330 714 NR1H3-1563-1629 26.54 9.20 331 715NR1H3-1564-1630 34.34 14.27 332 716 NR1H3-1565-1631 89.42 16.51 333 717NR1H3-1567-1633 66.56 5.80 334 718 NR1H3-1569-1635 39.41 11.96 335 719NR1H3-1570-1636 25.11 4.45 336 720 NR1H3-1572-1638 24.43 4.3 337 721NR1H3-1573-1639 13.36 2.16 338 722 NR1H3-1574-1640 82.95 11.20 339 723NR1H3-1577-1643 35.42 6.60 340 724 NR1H3-1579-1645 18.88 2.81 341 725NR1H3-1580-1646 21.56 5.67 342 726 NR1H3-1581-1647 4.90 1.52 343 727NR1H3-1582-1648 10.89 3.83 344 728 NR1H3-1583-1649 13.79 6.45 345 729NR1H3-1584-1650 16.62 7.10 346 730 NR1H3-1585-1651 5.99 2.91 347 731NR1H3-1586-1652 3.86 0.94 348 732 NR1H3-1587-1653 4.72 0.90 349 733NR1H3-1588-1654 2.35 0.39 350 734 NR1H3-1589-1655 47.09 6.4 351 735NR1H3-1590-1656 12.18 2.49 352 736 NR1H3-1591-1657 7.90 3.08 353 737NR1H3-1592-1658 13.57 3.13 354 738 NR1H3-1593-1659 31.19 18.86 355 739NR1H3-1656-1720 31.57 13.68 356 740 NR1H3-1657-1721 21.24 4.73 357 741NR1H3-1658-1722 15.81 4.89 358 742 NR1H3-1659-1723 12.33 4.79 359 743NR1H3-1660-1724 22.98 7.43 360 744 NR1H3-1661-1725 9.37 3.58 361 745NR1H3-1662-1726 8.29 2.01 362 746 NR1H3-1663-1727 4.60 1.56 363 747NR1H3-1664-1728 16.48 1.97 364 748 NR1H3-1665-1729 17.41 2.6 365 749NR1H3-1666-1730 23.86 4.49 366 750 NR1H3-1667-1731 23.67 5.80 367 751NR1H3-1668-1732 27.34 4.76 368 752 NR1H3-1669-1733 9.25 2.46 369 753NR1H3-1671-1735 5.99 1.88 370 754 NR1H3-1677-1741 28.16 4.53 371 755NR1H3-1679-1743 10.30 2.96 372 756 NR1H3-1680-1744 13.50 3.80 373 757NR1H3-1681-1745 12.10 2.74 374 758 NR1H3-1682-1746 17.59 5.47 375 759NR1H3-1683-1747 17.57 14.50 376 760 NR1H3-1684-1748 3.97 0.47 377 761NR1H3-1685-1749 7.55 2.88 378 762 NR1H3-1686-1750 8.97 4.18 379 763NR1H3-1687-1751 16.24 8.75 380 764 NR1H3-1728-1792 6.44 1.49 381 765NR1H3-1729-1793 8.96 3.52 382 766 NR1H3-1730-1794 15.41 8.06 383 767NR1H3-1731-1795 5.40 2.04 384 768 NR1H3-1732-1796 3.85 0.85

Example 3: GalNAc-Conjugated NR1H3 RNAi Oligonucleotides Inhibit HumanNR1H3 In Vitro

The in vitro screening assay in Example 2 validated the ability ofNTR1H3-targeting DsiRNA to knockdown NR1H3 mRNA. To further evaluate theability of NR1H3 RNAi oligonucleotides to inhibit NR1H3 mRNA expression,GalNAc-conjugated NR1H3 oligonucleotides were generated using sequencesidentified by the algorithm in Example 2.

Specifically, a subset of the DsiRNAs identified by the algorithm wereused to generate corresponding double-stranded RNAi oligonucleotidescomprising a nicked tetraloop GalNAc-conjugated structure (referred toherein as “GalNAc-conjugated NR1H3 oligonucleotides” or “GalNAc-NR1H3constructs”) having a 36-mer passenger strand and a 22-mer guide strand(Table 3). Further, two nucleotide sequences comprising the passengerstrand and guide strand have a distinct pattern of modified nucleotidesand phosphorothioate linkages (sense strand SEQ ID Nos: 945-1032;antisense SEQ ID Nos:1037 and 1086). Three of the nucleotides comprisingthe tetraloop were each conjugated to a GalNAc moiety (CAS #14131-60-3).The modification pattern of each strand is illustrated below:

Sense Strand: 5′-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX[-mX-]₁₆-[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]- mX-mX-mX-mX-mX-mX-3′.

Hybridized to:

Antisense Strand: 5′-[MePhosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S- mX-3′.(Modification key: Table 2 and [ademX-GalNAc]=GalNAc-conjugatednucleotide)Or, represented as:

Sense Strand: [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][adem A-GalNAc][adem A-GalNAc][adem A-GalNAc][mX][mX][mX][mX][mX][mX]

Hybridized to:

Antisense Strand: [MePhosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX] [mXs][mXs][mX]The remainder of the nucleotide sequences comprising the passengerstrand and guide strand have a second distinct pattern of modifiednucleotides and phosphorothioate linkages (sense strand SEQ ID Nos:945-1032; antisense SEQ ID NOs: 1033-1036, 1038-1085 and 1087-1120).Three of the nucleotides comprising the tetraloop were each conjugatedto a GalNAc moiety (CAS #14131-60-3). The modification pattern of eachstrand is illustrated below:

Sense Strand: 5′-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX[-mX-]₁₆-[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]- mX-mX-mX-mX-mX-mX-3′.

Hybridized to:

Antisense Strand: 5′-[MePhosphonate-4O-mX]-S-fX-S-fX-S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX- S-mX-3′.(Modification key: Table 2 and [ademX-GalNAc]=GalNAc-conjugatednucleotide)Or, represented as:

Sense Strand: [mXs][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][adem A-GalNAc][adem A-GalNAc][adem A-GalNAc][mX][mX][mX][mX][mX][mX]

Hybridized to:

Antisense Strand: [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX] [mX][mX][mXs][mXs][mX]

TABLE 2 (Modification key:). Symbol Modification/linkage Key 1 mX2′-O-methyl modified nucleotide fX 2′-fluoro modified nucleotide -S-phosphorothioate linkage - phosphodiester linkage [MePhosphonate-4O-mX]5′-methoxyphosphonate-4′-oxy modified nucleotide ademA-GalNAc GalNAcattached to an adenine nucleotide Key 2 [mXs] 2′-O-methyl modifiednucleotide with a phosphorothioate linkage to the neighboring nucleotide[fXs] 2′-fluoro modified nucleotide with a phosphorothioate linkage tothe neighboring nucleotide [mX] 2′-O-methyl modified nucleotide withphosphodiester linkages to neighboring nucleotides [fX] 2′-fluoromodified nucleotide with phosphodiester linkages to neighboringnucleotides

GalNAc-NR1H3 constructs were used to evaluate inhibition efficacy incells expressing human NR1H3. Specifically, Huh-7 cells were transfectedwith the GalNAc-NR1H3 constructs using methods described in Example 2.Results are provided in Table 3, which demonstrate successful knock-downof human NR1H3 mRNA with several constructs.

TABLE 3 GalNAc-Conjugated Human NR1H3 RNAi Oligonucleotides SED ID SEDID NO NO (modified (modified Anti- Sense sense % Strand) Strand) DsiRNAname remaining SEM 945 1033 NR1H3-763-783-860 109.29 17.94 946 1034NR1H3-765-785-862 57.30 15.34 947 1035 NR1H3-767-787-864 31.55 7.72 9481036 NR1H3-768-788-865 89.93 18.03 949 1037 NR1H3-769-789-866 18.84 4.39950 1038 NR1H3-794-814-891 40.26 12.66 951 1039 NR1H3-1152-1172-124974.17 12.97 952 1040 NR1H3-1189-1209-1286 47.09 11.14 953 1041NR1H3-1195-1215-1292 41.72 9.81 954 1042 NR1H3-1200-1220-1297 102.5025.27 955 1043 NR1H3-1201-1221-1298 28.78 4.63 956 1044NR1H3-1202-1222-1299 16.81 5.40 957 1045 NR1H3-1203-1223-1300 11.40 3.12958 1046 NR1H3-1204-1224-1301 10.27 9.33 959 1047 NR1H3-1205-1225-130215.93 5.10 960 1048 NR1H3-1206-1226-1303 59.82 14.86 962 1050NR1H3-1208-1228-1305 10.94 1.83 963 1051 NR1H3-1209-1229-1306 101.6223.09 964 1052 NR1H3-1210-1230-1307 25.85 6.52 965 1053NR1H3-1211-1231-1308 29.68 4.19 966 1054 NR1H3-1212-1232-1309 39.9013.07 967 1055 NR1H3-1213-1233-1310 15.16 4.91 968 1056NR1H3-1214-1234-1311 25.26 16.03 970 1058 NR1H3-1221-1241-1318 23.776.65 973 1061 NR1H3-1249-1269-1346 45.75 8.54 975 1063NR1H3-1254-1274-1351 36.79 10.59 976 1064 NR1H3-1255-1275-1352 16.794.54 977 1065 NR1H3-1256-1276-1353 55.93 16.15 978 1066NR1H3-1257-1277-1354 90.17 15.01 980 1068 NR1H3-1260-1280-1357 17.802.89 981 1069 NR1H3-1261-1281-1358 81.65 24.27 982 1070NR1H3-1263-1283-1360 15.12 5.37 983 1071 NR1H3-1264-1284-1361 25.69 6.43986 1074 NR1H3-1405-1471-1502 40.51 10.03 987 1075 NR1H3-1409-1475-150643.45 7.797 993 1081 NR1H3-1458-1524-1555 6.54 1.345 995 1083NR1H3-1464-1530-1561 53.64 10.29 996 1084 NR1H3-1465-1531-1562 33.7810.83 997 1085 NR1H3-1467-1533-1564 33.29 19.93 998 1086NR1H3-1469-1535-1566 20.17 7.60 999 1087 NR1H3-1470-1536-1567 34.02 6.951002 1088 NR1H3-1480-1546-1577 31.73 6.53 1004 1092 NR1H3-1482-1548-157912.18 3.56 1009 1097 NR1H3-1524-1590-1621 28.36 10.49 1018 1106NR1H3-1594-1660-1691 30.67 13.85 1019 1107 NR1H3-1595-1661-1692 50.808.90 1020 1108 NR1H3-1596-1662 56.02 19.49 1022 1110 NR1H3-1670-173410.71 5.09 1024 1112 NR1H3-1672-1736 25.94 11.33 1025 1113NR1H3-1673-1737 53.70 17.23 1026 1114 NR1H3-1674-1738 14.48 2.82 10271115 NR1H3-1675-1739-1766 55.04 10.59 1028 1116 NR1H3-1676-1740-176733.47 12.90 1029 1117 NR1H3-1678-1742-1769 364.22 31.30

Example 4: RNAi Oligonucleotide Inhibition of NR1H3 Expression in MiceIn Vivo

The in vitro screening assays in Examples 2 and 3 validated the abilityof NR1H3-targeting oligonucleotides to knock-down target mRNA. Toconfirm the ability of the RNAi oligonucleotides to knockdown NR1H3 invivo, an HDI mouse model was used. Two sets of GalNAc-conjugated NR1H3oligonucleotides were evaluated. Specifically, a set ofGalNAc-conjugated NR1H3 oligonucleotides were generated from DsiRNAscreened in Example 2 (as shown in Table 4) and the GalNAc-conjugatedNR1H3 oligonucleotides from Example 3 (as shown in Table 5). Both setsof constructs were evaluated in mice engineered to transiently expresshuman NR1H3 mRNA in hepatocytes of the mouse liver. Briefly,6-8-week-old female CD-1 mice (n=4-5) were subcutaneously administeredthe indicated GalNAc-conjugated NR1H3 oligonucleotides at a dose of 2mg/kg formulated in PBS. A control group of mice (n=5) were administeredonly PBS. Three days later (72 hours), the mice were hydrodynamicallyinjected (HDI) with a DNA plasmid encoding the full human NR1H3 gene (25μg) under control of a ubiquitous cytomegalovirus (CMV) promotersequence. One day after introduction of the DNA plasmid, liver samplesfrom HDI mice were collected. Total RNA derived from these HDI mice weresubjected to qRT-PCR analysis to determine NR1H3 mRNA levels asdescribed in Example 2. mRNA levels were measured for both human andmouse mRNA. The values were normalized for transfection efficiency usingthe NeoR gene included on the DNA plasmid.

TABLE 4 GalNAc-Conjugated Human NR1H3 RNAi Oligonucleotides for HDIscreen Unmodified Unmodified Modified Modified Sense Strand AntisenseSense Strand Antisense (SED ID strand (SED ID strand NO) (SED ID NO) NO)(SED ID NO) NR1H3-1207- 823 911 961 1049 1227 NR1H3-1220- 824 912 9691057 1240 NR1H3-1224- 825 913 971 1059 1244 NR1H3-1244- 826 914 972 10601264 NR1H3-1252- 827 915 974 1062 1272 NR1H3-1259- 828 916 979 1067 1279NR1H3-1265- 829 917 984 1072 1285 NR1H3-1266- 830 918 985 1073 1286NR1H3-1429- 831 919 988 1076 1495 NR1H3-1433- 832 920 989 1077 1499NR1H3-1437- 833 921 990 1078 1503 NR1H3-1439- 834 922 991 1079 1505NR1H3-1446- 835 923 992 1080 1512 NR1H3-1463- 836 924 994 1082 1529NR1H3-1475- 837 925 1000 1088 1541 NR1H3-1479- 838 926 1001 1089 1545NR1H3-1481- 839 927 1003 1091 1547 NR1H3-1485- 840 928 1005 1093 1551NR1H3-1515- 1537 929 1006 1094 1581 NR1H3-1517- 842 930 1007 1095 1583NR1H3-1518- 843 931 1008 1096 1584 NR1H3-1533- 844 932 1010 1098 1599NR1H3-1535- 845 933 1011 1099 1601 NR1H3-1545- 846 934 1012 1100 1611NR1H3-1554- 847 935 1013 1101 1620 NR1H3-1581- 848 936 1014 1102 1647NR1H3-1586- 849 937 1015 1103 1652 NR1H3-1587- 850 938 1016 1104 1653NR1H3-1588- 851 939 1017 1105 1654 NR1H3-1663- 852 940 1021 1109 1727NR1H3-1671- 853 941 1023 1111 1735 NR1H3-1684- 854 942 1030 1118 1748NR1H3-1731- 855 943 1031 1119 1795 NR1H3-1732- 856 944 1032 1120 1796

TABLE 5 GalNAc-Conjugated Human NR1H3 RNAi Oligonucleotides for HDIscreen Unmodified Unmodified Modified Modified Sense Strand AntisenseSense Strand Antisense (SED ID strand (SED ID strand NO) (SED ID NO) NO)(SED ID NO) NR1H3-763- 769 857 945 1033 783-860 NR1H3-765- 770 858 9461034 785-862 NR1H3-767- 771 859 947 1035 787-864 NR1H3-768- 772 860 9481036 788-865 NR1H3-769- 773 861 949 1037 789-866 NR1H3-794- 774 862 9501038 814-891 NR1H3-1152- 775 863 951 1039 1172-1249 NR1H3-1189- 776 864952 1040 1209-1286 NR1H3-1195- 777 865 953 1041 1215-1292 NR1H3-1200-778 866 954 1042 1220-1297 NR1H3-1201- 779 867 955 1043 1221-1298NR1H3-1202- 780 868 956 1044 1222-1299 NR1H3-1203- 781 869 957 10451223-1300 NR1H3-1204- 782 870 958 1046 1224-1301 NR1H3-1205- 783 871 9591047 1225-1302 NR1H3-1206- 784 872 960 1048 1226-1303 NR1H3-1208- 785873 962 1050 1228-1305 NR1H3-1209- 786 874 963 1051 1229-1306NR1H3-1210- 787 875 964 1052 1230-1307 NR1H3-1211- 788 876 965 10531231-1308 NR1H3-1212- 789 877 966 1054 1232-1309 NR1H3-1213- 790 878 9671055 1233-1310 NR1H3-1214- 791 879 968 1056 1234-1311 NR1H3-1221- 792880 970 1058 1241-1318 NR1H3-1249- 793 881 973 1061 1269-1346NR1H3-1254- 794 882 975 1063 1274-1351 NR1H3-1255- 795 883 976 10641275-1352 NR1H3-1256- 796 884 977 1065 1276-1353 NR1H3-1257- 797 885 9781066 1277-1354 NR1H3-1260- 798 886 980 1068 1280-1357 NR1H3-1261- 799887 981 1069 1281-1358 NR1H3-1263- 800 888 982 1070 1283-1360NR1H3-1264- 801 889 983 1071 1284-1361 NR1H3-1405- 802 890 986 10741471-1502 NR1H3-1409- 803 891 987 1075 1475-1506 NR1H3-1458- 804 892 9931081 1524-1555 NR1H3-1464- 805 893 995 1083 1530-1561 NR1H3-1465- 806894 996 1084 1531-1562 NR1H3-1467- 807 895 997 1085 1533-1564NR1H3-1469- 808 896 998 1086 1535-1566 NR1H3-1470- 809 897 999 10871536-1567 NR1H3-1480- 810 898 1002 1090 1546-1577 NR1H3-1482- 811 8991004 1092 1548-1579 NR1H3-1524- 812 900 1009 1097 1590-1621 NR1H3-1594-813 901 1018 1106 1660-1691 NR1H3-1595- 814 902 1019 1107 1661-1692NR1H3-1596- 815 903 1020 1108 1662 NR1H3-1670- 816 904 1022 1110 1734NR1H3-1672- 817 905 1024 1112 1736 NR1H3-1673- 818 906 1025 1113 1737NR1H3-1674- 819 907 1026 1114 1738 NR1H3-1675- 820 908 1027 11151739-1766 NR1H3-1676- 821 909 1028 1116 1740-1767 NR1H3-1678- 822 9101029 1117 1742-1769

The results in FIG. 1B and FIG. 2 demonstrate that GalNAc-conjugatedNR1H3 oligonucleotides designed to target human NR1H3 mRNA successfullyinhibited human NR1H3 mRNA expression in HDI mice, as determined by areduction in the amount of human NR1H3 mRNA expression in liver samplesfrom HDI mice treated with GalNAc-conjugated NR1H3 oligonucleotidesrelative to control HDI mice treated with only PBS. Benchmark controls(NR1H3-769 and NR1H3-1469), which were selected from a prior sequencescreening, were used to confirm successful knock-down.

The GalNAc-conjugated NR1H3 oligonucleotides tested in FIG. 1B and FIG.2 were further validated in repeat assays as shown in FIGS. 3 and 4using constructs from Table 4 and Table 5. The assays verifiedknock-down efficiency of each GalNAc-conjugated NR1H3 oligonucleotide,and four constructs were selected for further analysis in non-humanprimates (NHP).

Example 5: RNAi Oligonucleotide Inhibition of NR1H3 Expression inNon-Human Primates In Vivo

Effective GalNAc-NR1H3 constructs identified in the HDI mouse studieswere assayed for targeting efficiency in non-human primates.Specifically, GalNAc-conjugated NR1H3 oligonucleotides listed in Table 6were evaluated in non-naïve cynomolgus monkeys (Macaca fascicularis).

TABLE 6 GalNAc Constructs evaluated in non-human primate Modified SenseModified strand Antisense strand (SED ID NO) (SED ID NO)NR1H3-1209-1229- 963 1051 1306 NR1H3-1210-1230- 964 1052 1307NR1H3-1515-1581 1006 1094 NR1H3-1594-1660- 1018 1106 1691

In this study, the monkeys were grouped so that their mean body weights(about 5.4 kg) were comparable between the control and experimentalgroups. Each cohort contained at least two female and at least two malesubjects. The GalNAc-conjugated NR1H3 oligonucleotides were administeredsubcutaneously at a dose of 1 or 4 mg/kg on Study Day 0, 28, 56, and 112as outlined in FIG. 5 . Blood samples and liver biopsies were collectedas outlined in Table 7 and FIG. 5 .

TABLE 7 Treatment plan for NHP study Dose DP Blood Liver Group (mg/kg)Dosing GalXC Number Collection Biopsy N A N/A sc PBS N/A −8, 0, 14, −8,28, 56, 5 28, 56, 112 112 B 1 sc NR1H3-1209- DP18987P: −8, 0, 14, 28,56, 112 5 1229-1306 DP18986G 28, 56, 112 C 4 sc NR1H3-1209- DP18987P:−8, 0, 14, −8, 28, 56, 5 1229-1306 DP18986G 28, 56, 112 112 D 1 scNR1H3-1210- DP18989P: −8, 0, 14, 28, 56, 112 5 1230-1307 DP18988G 28,56, 112 E 4 sc NR1H3-1210- DP18989P: −8, 0, 14, −8, 28, 56, 5 1230-1307DP18988G 28, 56, 112 112 F 1 sc NR1H3-1515- DP20645P: −8, 0, 14, 28, 56,112 5 1581 DP20644G 28, 56, 112 G 4 sc NR1H3-1515- DP20645P: −8, 0, 14,−8, 28, 56, 5 1581 DP20644G 28, 56, 112 112 H 1 sc NR1H3-1594- DP19040P:−8, 0, 14, 28, 56, 112 4 1660-1691 DP19039G 28, 56, 112 I 4 scNR1H3-1594- DP19040P: −8, 0, 14, −8, 28, 56, 5 1660-1691 DP19039G 28,56, 112 112

At each time point, total RNA derived from the liver biopsy samples wassubjected to qRT-PCR analysis to measure NR1H3 mRNA inoligonucleotide-treated monkeys relative to those treated with acomparable volume of PBS. To normalize the data, the measurements weremade relative to the reference gene, PPIB (Rh02802984_m1(Tagman)). Thefollowing SYBR assays purchased from Integrated DNA Technologies wereused to evaluate gene expressions: Forward-942: GTCTCTGTGCAGGAGATAGTTG(SEQ ID NO: 1517), Reverse-1399: GGAGGCTCACCAGTTTCATTA (SEQ ID NO:1518). As shown in Table 8 (Day 28), treating NHPs with theGalNAc-conjugated NR1H3 oligonucleotides listed in Table 6 inhibitedNR1H3 expression in the liver, as determined by a reduced amount ofNR1H3 mRNA in liver samples from oligonucleotide-treated NHPs relativeto NHPs treated with PBS. Days 56 and 112 were also measured (Table 8).These results demonstrate that treating NHPs with the GalNAc-conjugatedNR1H3 oligonucleotides reduces the amount of NR1H3 mRNA in the liver ina dose dependent manner.

TABLE 8 NR1H3 mRNA remaining after treatment with the indicatedNR1H3-GalNAc in the liver from the oligonucleotide-treated NHP NR1H3Gene Expression (% Time-Matched PBS) −1209 −1210 −1515 −1594 PBS 1 mg/kg4 mg/kg 1 mg/kg 4 mg/kg 1 mg/kg 4 mg/kg 1 mg/kg 4 mg/kg Day 28 Animal 1107.3 58.6 35.6 60.1 45.1 95.8 55.4 111.6 80.4 Animal 2 116.6 62.4 27.848.5 34.7 101.3 NS 108.3 103.6 Animal 3 122.1 79.4 44.5 73.8 35.7 111.874.1 145.5 45.9 Animal 4 77.4 78.5 50.5 65.1 27.5 70.0 45.1 195.4 73.2Animal 5 76.6 89.2 37.4 77.5 45.2 78.9 34.9 NS 68.5 Average 100.0 73.639.2 65.0 37.6 91.6 52.4 140.2 74.3 SEM 9.7 5.7 3.9 5.1 3.4 7.6 8.4 20.29.3 Day 56 Animal 1 96.7 72.9 29.0 43.7 39.0 59.7 30.0 67.7 78.2 Animal2 115.0 60.1 29.7 75.9 32.1 68.7 NS 85.1 103.4 Animal 3 87.1 52.7 32.636.8 40.4 48.1 45.0 104.3 70.0 Animal 4 103.4 34.9 45.0 51.9 26.9 43.637.3 76.2 72.1 Animal 5 97.8 45.9 32.3 55.9 36.0 47.1 31.1 NS 72.4Average 100.0 53.3 33.7 52.8 34.9 53.4 35.9 83.3 79.2 SEM 4.6 6.4 2.96.6 2.5 4.7 3.4 7.8 6.2 Day 112 Animal 1 113.2 44.2 27.3 34.4 33.1 52.846.8 56.6 34.8 Animal 2 106.6 56.2 21.7 42.8 33.0 36.2 NS 57.9 31.2Animal 3 100.2 59.6 22.1 33.4 29.6 46.2 45.7 48.5 44.2 Animal 4 103.147.8 45.0 44.9 33.1 44.1 44.9 65.7 32.1 Animal 5 77.0 55.9 34.4 45.332.1 52.6 37.6 NS 33.6 Average 100.0 52.7 30.1 40.2 32.2 46.4 43.8 57.235.2 SEM 6.2 2.9 4.4 2.6 0.7 3.1 2.1 3.5 2.3 NS = no sample

To confirm if the GalNAc conjugated NR1H3 were specific targeting NR1H3,both NR1H3 and NR1H2 expression was evaluated in liver samples.Non-human primates treated with 4 mg/kg of GalNAc-conjugated NR1H3 wereassayed for expression at days 0, 28, 56, and 112 in the liver forknock-down (KD) of NR1H3 mRNA in (Table 9), and NR1H2 (Table 10). Theresults demonstrate that the constructs are specific to NR1H3 and do nothave significant off target effects for NR1H2.

TABLE 9 NR1H3 mRNA remaining after treatment with the indicatedNR1H3-GalNAc in NHP NR1H3 Gene Expression (% Time Matched PBS) 4 mg/kgPBS −1209 −1210 −1515 −1594 Day −6 Animal 1 65.8 136.6 204.3 103.8 NSAnimal 2 102.4 78.1 113.3 49.3 80.3 Animal 3 86.1 101.0 150.2 94.0 104.3Animal 4 137.3 89.9 155.0 82.8 109.7 Animal 5 108.4 90.8 92.5 83.1 124.2Average 100.0 99.3 143.1 82.6 104.6 SEM 11.9 10.0 19.2 9.2 9.1 Day 28Animal 1 107.3 35.6 45.1 55.4 80.4 Animal 2 116.6 27.8 34.7 NS 103.6Animal 3 122.1 44.5 35.7 74.1 45.9 Animal 4 77.4 50.5 27.5 45.1 73.2Animal 5 76.6 37.4 45.2 34.9 68.5 Average 100.0 39.2 37.6 52.4 74.3 SEM9.7 3.9 3.4 8.4 9.3 Day 56 Animal 1 96.7 29.0 39.0 30.0 78.2 Animal 2115.0 29.7 32.1 NS 103.4 Animal 3 87.1 32.6 40.4 45.0 70.0 Animal 4103.4 45.0 26.9 37.3 72.1 Animal 5 97.8 32.3 36.0 31.1 72.4 Average100.0 33.7 34.9 35.9 79.2 SEM 4.6 2.9 2.5 3.4 6.2 Day 112 Animal 1 113.227.3 33.1 46.8 34.8 Animal 2 106.6 21.7 33.0 NS 31.2 Animal 3 100.2 22.129.6 45.7 44.2 Animal 4 103.1 45.0 33.1 44.9 32.1 Animal 5 77.0 34.432.1 37.6 33.6 Average 100.0 30.1 32.2 43.8 35.2 SEM 6.2 4.4 0.7 2.1 2.3NS = no sample

TABLE 10 NR1H2 mRNA remaining after treatment with the indicatedNR1H3-GalNAc in NHP NR1H2 Gene Expression (% Time Matched PBS) 4 mg/kgPBS −1209 −1210 −1515 −1594 Day −6 Animal 1 119.7 84 324.9 79.8 NSAnimal 2 96.3 107.5 116.1 89.3 175.9 Animal 3 105.7 86.2 106.6 103 151.1Animal 4 82.1 105.7 672 127.9 113.2 Animal 5 96.3 134.9 126.7 101.6113.9 Average 100.0 103.7 269.3 100.3 138.5 SEM 6.2 9.2 108.5 8.1 15.3Day 28 Animal 1 101 80.2 122.2 102.7 117.5 Animal 2 123.3 80.9 103.7 NS130 Animal 3 111.8 78.1 107.3 87.8 61.2 Animal 4 88.5 120.4 113.7 98.380.2 Animal 5 75.3 129.4 102.7 97.1 75.4 Average 100.0 97.8 109.9 96.592.9 SEM 8.4 11.2 3.6 3.1 13.1 Day 56 Animal 1 118.5 127.9 119.2 90.988.5 Animal 2 118.4 98.6 99.6 NS 112 Animal 3 90.9 91 131.8 76.3 103Animal 4 88.5 115.4 117.8 100.7 121.1 Animal 5 83.8 107.6 87.1 85.5119.4 Average 100.0 108.1 111.1 88.4 108.8 SEM 7.6 6.4 7.9 5.1 6.0 Day112 Animal 1 105.6 114.2 107.9 95.6 89.6 Animal 2 108.5 81.5 106.8 NS80.8 Animal 3 119.1 76.7 118.4 90.5 96.8 Animal 4 100.4 121.1 97.2 95.498.6 Animal 5 66.4 99.1 104.8 111.9 98.1 Average 100.0 98.5 107.0 98.492.8 SEM 8.9 8.7 3.4 4.7 3.4

Taken together, these results show that GalNAc-conjugated NR1H3oligonucleotides designed to target human NR1H3 mRNA inhibit total NR1H3expression in vivo (as determined by the reduction of the amount ofNR1H3 mRNA).

Example 6

In order to determine the effect of hepatic NR1H3 knockdown on liver andplasma triglycerides and total plasma cholesterol, C57B1 mice fed a highfat, high fructose, high cholesterol (HFFC-NASH) diet were administrateda NR1H3 GalNac siRNA probe specifically designed to reduce NR1H3expression in mice. The compound was administrated by subcutaneousinjection 3 mg/kg once a week for 4 weeks. The respective vehicletreated groups (chow and HFFC-NASH diet) were treated with PBS.

As seen in Table 11, hepatocyte specific knockdown (KD) of murineNR1H3-specific GalNac siRNA led to a significant decrease in murineNR1H3 transcript expression in the liver of treated animals as comparedto vehicle. Treatment leading to such hepatic NR1H3 mRNA reduction inturn induced reductions in liver triglycerides, plasma triglyceride andplasma total cholesterol. Furthermore, genes involved in hepatic de novolipogenesis (fatty acid synthase (Fasn) and Acetyl-CoA carboxylase 2(Acc2)) were decreased by treatment with the murine NR1H3-specificGalNac siRNA. In conclusion, hepatocyte-specific knockdown of NR1H3 mRNAin a mouse model for NAFLD led to significant improvement in NASHrelated dyslipidemia.

TABLE 11 NASH p-value diet/NR1H3 NASH/ CHOW diet NASH KD NASH AVG ± ST.AVG ± ST. AVG ± ST. NR1H3 DEV. DEV. DEV. KD Liver 9.39 ± 2.88 47.00 ±12.56 19.39 ± 11.07 <0.0001 triglycerides mg/g liver Plasma 1.31 ± 0.310.86 ± 0.21 0.46 ± 0.11  0.0036 triglycerides mM Plasma total 2.06 ±0.21 4.19 ± 0.54 3.37 ± 0.67  0.0145 cholesterol mM NR1H3  100 ± 5.27106.19 ± 8.75  43.88 ± 6.27  <0.0001 mRNA expression Fasn mRNA   100 ±44.53 56.60 ± 27.23 17.58 ± 6.06   0.0327 expression Acc2 mRNA   100 ±20.30 43.34 ± 21.89 12.21 ± 4.31   0.0034 expression

Example 7: GalNAc-Conjugated siRNA Targeting NR1H3 for Knock Down inObese Rhesus Monkeys Fed a High Cholesterol Diet

A GalNAc-conjugated NR1H3 oligonucleotide (sense strand SEQ ID NO: 964antisense strand SEQ ID NO: 1052) was administered to obese rhesusmonkeys fed a high cholesterol diet (0.06% cholesterol). Eight obeserhesus monkeys fed a high cholesterol diet (0.06% cholesterol) for morethan 4 years, and with a body weight ranging from 8-25 kg, were dosedwith 4 mg/kg lead at weeks 0, 4 and 8 and study was terminated at week12. A liver biopsy (week −3) and an ultrasound scan of the liver (week−1) were performed prior to dosing of the animals. Liver enzymes andplasma lipids were determined prior to dosing and every 2 weeksposttreatment to assess potential adverse effects. At the end of thestudy, liver and plasma samples were collected to measure the effect ofNR1H3a knock down on lipid and cholesterol metabolism, hepatic genesinvolved in these pathways, as well as liver histology.

At the end of the study NR1H3a mRNA in the liver was decreased 50%. mRNAis calculated as Δ/Δ Ct levels relative to 18s gene expression in eachsample. As NR1H3a is expressed in other liver cells, e.g. the Kupffercells, a full knockdown of NR1H3a was not expected from a liverhomogenate.

The plasma levels of liver enzymes and plasma levels of lipids arepresented in Table 12. During the treatment period, there was no changein the plasma level of alanine aminotransferase (ALT),gamma-glutamyltransferase (GGT) or total bilirubin (TB), while a smallincrease was observed in aspartate aminotransferase (AST) after 8 and 10weeks of treatment. During the treatment period, there was no change inthe plasma level total cholesterol (TC) or low-density lipoproteincholesterol (LDL C), while a significant increase in high-densitylipoprotein cholesterol (HDL C) was observed already after 2 weeks oftreatment. After 12 weeks of treatment a significant decrease in plasmatriglycerides (TG) was observed. In Table 12, data representmean±standard error of the mean. Baseline is the average of measuresfrom week −4 to week 0 (pre-dose). Statistics are done relative tobaseline: One-Way ANOVA. P*<0.05 P**<0.01, P***<0.001.

TABLE 12 Liver enzymes and plasma lipids in response to NR1H3a KD inobese rhesus monkeys. Plasma Plasma Total AST ALT GGT TBili TGCholesterol HDL-C LDL-C (IU/L) (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL)(mg/dL) (mg/dL) Baseline 31.9 ± 1.5    55.6 ± 7.1 55.1 ± 4.2 0.21 ±0.01  110 ± 19   206 ± 21.9 80 ± 4.6   107 ± 17.3 Week 2  32.9 ± 2.9   58.4 ± 6.2 55.6 ± 5.2 0.17 ± 0.03  84.9 ± 10.3  209 ± 20  98 ± 6.0**  98± 14.5 Week 4  36.6 ± 4.1    68.9 ± 9.3 58.0 ± 3.8 0.18 ± 0.02   103 ±20.2  210 ± 15  107 ± 4.3*** 101 ± 10.8 Week 6  38.5 ± 3.2    66.3 ± 6.049.9 ± 3.2 0.23 ± 0.02  74.1 ± 6.7   208 ± 16  102 ± 6.4**  98 ± 9.8Week 8  42.5 ± 2.5*** 70.4 ± 9.0 54.1 ± 4.5 0.15 ± 0.02* 93.3 ± 18.0 211 ± 14  105 ± 6.2**  104 ± 9.4  Week 10 39.5 ± 2.8**  58.5 ± 3.7 60.0± 3.3 0.17 ± 0.02  91.6 ± 12.4  219 ± 15  107 ± 4.9**  105 ± 10.4 Week12 36.1 ± 2.1    59.3 ± 7.0 62.8 ± 3.5 0.16 ± 0.02* 84.9 ± 17.2* 214 ±14  108 ± 4.3**  116 ± 11.1

The effect of NR1H3a knock down on liver triglycerides and livercholesterol is presented in Table 13. No significant change in livertriglycerides or cholesterol was observed. However, a tendency (p=0.055,paired t-test) towards a decrease in liver stiffness as measured byFibroScan™ was observed in response to hepatic NR1H3a knock down. InTable 13 data represent mean±standard error of the mean analyzed by apaired t-test. One monkey was excluded from the FibroScan™ analysis asit was not fully sedated during the second scan.

TABLE 13 TG and cholesterol levels and FibroScan ™ liver stiffness inobese rhesus monkeys Baseline Week 13-14 Liver triglycerides (mg/dL)40.9 ± 6.6 40.0 ± 5.5 Liver cholesterol (mg/dL) 42.6 ± 3.9 41.4 ± 3.0Liver stiffness kPa  5.7 ± 0.5  4.3 ± 0.2

From the study, it can be concluded that knockdown of NR1H3a inhepatocytes does not cause hepatic cholesterol accumulation in obesemonkeys fed a high cholesterol diet. An increased in plasma HDL C wasobserved 2 weeks after dosing, while a lowering of plasma TG wasobserved at week 12. No effect on liver TG was observed but none of themonkeys had steatosis at the start of the study. However, a tendencytoward a decrease in liver stiffness was observed in response to hepaticNR1H3a knockdown.

LIST OF EMBODIMENTS

Embodiment 1. An RNAi oligonucleotide for reducing NR1H3 expression, theoligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein the antisense strand comprises a region of complementarity to aNR1H3 mRNA target sequence of any one of SEQ ID NOs: 1-384, and whereinthe region of complementarity is at least 15 contiguous nucleotides inlength.Embodiment 2. The RNAi oligonucleotide of embodiment 1, wherein thesense strand is 15 to 50 nucleotides in length.Embodiment 3. The RNAi oligonucleotide of embodiments 1 or 2, whereinthe sense strand is 18 to 36 nucleotides in length.Embodiment 4. The RNAi oligonucleotide of any one of embodiments 1 to 3,wherein the antisense strand is 15 to 30 nucleotides in length.Embodiment 5. The RNAi oligonucleotide of any one of embodiments 1 to 4,wherein the antisense strand is 22 nucleotides in length and whereinantisense strand and the sense strand form a duplex region of at least19 nucleotides in length, optionally at least 20 nucleotides in length.Embodiment 6. The RNAi oligonucleotide of any one of embodiments 1 to 5,wherein the region of complementarity is at least 19 contiguousnucleotides in length, optionally at least 20 nucleotides in length.Embodiment 7. The RNAi oligonucleotide of any one of embodiments 1 to 6,wherein the 3′ end of the sense strand comprises a stem-loop set forthas S1-L-S2, wherein S1 is complementary to S2, and wherein L forms aloop between S1 and S2 of 3-5 nucleotides in length.Embodiment 8. An RNAi oligonucleotide for reducing NR1H3 expression, theoligonucleotide comprising a sense strand of 15 to 50 nucleotides inlength and an antisense strand, wherein the sense strand and theantisense strand form a duplex region, wherein the wherein the antisensestrand comprises a region of complementarity to a NR1H3 mRNA targetsequence of any one of SEQ ID NOs: 1-384, and wherein the region ofcomplementarity is at least 15 contiguous nucleotides in length.Embodiment 9. An RNAi oligonucleotide for reducing NR1H3 expression, theoligonucleotide comprising a sense strand of 15 to 50 nucleotides inlength and an antisense strand of 15 to 30 nucleotides in length,wherein the sense strand and the antisense strand form a duplex region,wherein the antisense strand comprises a region of complementarity to aNR1H3 mRNA target sequence of any one of SEQ ID NOs: 1-384, and whereinthe region of complementarity is at least 15 contiguous nucleotides inlength.Embodiment 10. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 15 to 50 nucleotides inlength and an antisense strand, wherein the sense strand and theantisense strand form a duplex region, wherein the antisense strandcomprises a region of complementarity to a NR1H3 mRNA target sequence ofany one of SEQ ID NOs: 1-384, and wherein the region of complementarityis 19 contiguous nucleotides in length, optionally 20 nucleotides inlength.Embodiment 11. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 18 to 36 nucleotides inlength and an antisense strand, wherein the sense strand and theantisense strand form a duplex region, wherein the antisense strandcomprises a region of complementarity to a NR1H3 mRNA target sequence ofany one of SEQ ID NOs: 1-384, and wherein the region of complementarityis 19 contiguous nucleotides in length, optionally 20 nucleotides inlength.Embodiment 12. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 18 to 36 nucleotides inlength and an antisense strand of 22 nucleotides in length, wherein thesense strand and the antisense strand form a duplex region, wherein theantisense strand comprises a region of complementarity to a NR1H3 mRNAtarget sequence of any one of SEQ ID NOs: 1-384, and wherein the regionof complementarity is 19 contiguous nucleotides in length, optionally 20nucleotides in length.Embodiment 13. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 18 to 36 nucleotides inlength and an antisense strand of 22 nucleotides in length, wherein thesense strand and the antisense strand form a duplex region, wherein the3′ end of the sense strand comprises a stem-loop set forth as S1-L-S2,wherein S1 is complementary to S2, and wherein L forms a loop between S1and S2 of 3-5 nucleotides in length, wherein the antisense strandcomprises a region of complementarity to a NR1H3 mRNA target sequence ofany one of SEQ ID NOs: 1-384, and wherein the region of complementarityis 19 contiguous nucleotides in length, optionally 20 nucleotides inlength.Embodiment 14. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 36 nucleotides inlength and an antisense strand of 22 nucleotides in length, wherein thesense strand and the antisense strand form a duplex region, wherein the3′ end of the sense strand comprises a stem-loop set forth as S1-L-S2,wherein S1 is complementary to S2, and wherein L forms a loop between S1and S2 of 3-5 nucleotides in length, wherein the antisense strandcomprises a region of complementarity to a NR1H3 mRNA target sequence ofany one of SEQ ID NOs: 1-384, and wherein the region of complementarityis 19 contiguous nucleotides in length, optionally 20 nucleotides inlength.Embodiment 15. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand of 36 nucleotides inlength and an antisense strand of 22 nucleotides in length, wherein thesense strand and the antisense strand form a duplex region of at least19 nucleotides in length, optionally 20 nucleotides in length, whereinthe 3′ end of the sense strand comprises a stem-loop set forth asS1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loopbetween S1 and S2 of 3-5 nucleotides in length, wherein the antisensestrand comprises a region of complementarity to a NR1H3 mRNA targetsequence of any one of SEQ ID NOs: 1-384, and wherein the region ofcomplementarity is 19 contiguous nucleotides in length, optionally 20nucleotides in length.Embodiment 16. The RNAi oligonucleotide of any one of embodiments 7 and13-15, wherein L is a triloop or a tetraloop.Embodiment 17. The RNAi oligonucleotide of embodiment 16, wherein L is atetraloop.Embodiment 18. The RNAi oligonucleotide of embodiment 17, wherein thetetraloop comprises the sequence 5′-GAAA-3′.Embodiment 19. The RNAi oligonucleotide of any one of embodiments 16-18,wherein the S1 and S2 are 1-10 nucleotides in length and have the samelength.Embodiment 20. The RNAi oligonucleotide of embodiment 19, wherein S1 andS2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides,or 10 nucleotides in length.Embodiment 21. The RNAi oligonucleotide of embodiment 20, wherein S1 andS2 are 6 nucleotides in length.Embodiment 22. The RNAi oligonucleotide of any one of embodiments 16 to21, wherein the stem-loop comprises the sequence 5′-GCAGCCGAAAGGCUGC-3′(SEQ ID NO: 1121).Embodiment 23. The RNAi oligonucleotide of any one of embodiments 1 to22, wherein the antisense strand comprises a 3′-overhang sequence of oneor more nucleotides in length.Embodiment 24. The RNAi oligonucleotide of embodiment 23, wherein the3′-overhang sequence is 2 nucleotides in length, optionally wherein the3′-overhang sequence is GG.Embodiment 25. The RNAi oligonucleotide of any one of the precedingembodiments, wherein the oligonucleotide comprises at least one modifiednucleotide.Embodiment 26. The RNAi oligonucleotide of embodiment 25, wherein themodified nucleotide comprises a 2′-modification.Embodiment 27. The RNAi oligonucleotide of embodiment 26, wherein the2′-modification is a modification selected from 2′-aminoethyl,2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.Embodiment 28. The RNAi oligonucleotide of any one of embodiments 25 to27, wherein all nucleotides comprising the oligonucleotide are modified,optionally wherein the modification is a 2′-modification selected from2′-fluoro and 2′-O-methyl.Embodiment 29. The RNAi oligonucleotide of any one of embodiments 25-28,wherein about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotidesof the sense strand comprise a 2′-fluoro modification.Embodiment 30. The RNAi oligonucleotide of any one of embodiments 25-29,wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,or 35% of the nucleotides of the antisense strand comprise a 2′-fluoromodification.Embodiment 31. The RNAi oligonucleotide of any one of embodiments 25-30,wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,or 35% of the nucleotides of the oligonucleotide comprise a 2′-fluoromodificationEmbodiment 32. The RNAi oligonucleotide of any one of embodiments 25-31,wherein the sense strand comprises 36 nucleotides with positions 1-36from 5′ to 3′, wherein positions 8-11 comprise a 2′-fluoro modification.Embodiment 33. The RNAi oligonucleotide of any one of embodiments 25-32,wherein the antisense strand comprises 22 nucleotides with positions1-22 from 5′ to 3′, and wherein positions 2, 3, 4, 5, 7, 10, and 14comprise a 2′-fluoro modification.Embodiment 34. The RNAi oligonucleotide of any one of embodiments 25-33,wherein the remaining nucleotides comprise a 2′-O-methyl modification.Embodiment 35. The RNAi oligonucleotide of any one of the precedingembodiments, wherein the oligonucleotide comprises at least one modifiedinternucleotide linkage.Embodiment 36. The RNAi oligonucleotide of embodiment 35, wherein the atleast one modified internucleotide linkage is a phosphorothioatelinkage.Embodiment 37. The RNAi oligonucleotide of any one of the precedingembodiments, wherein the 4′-carbon of the sugar of the 5′-nucleotide ofthe antisense strand comprises a phosphate analog.Embodiment 38. The RNAi oligonucleotide of embodiment 37, wherein thephosphate analog is oxymethylphosphonate, vinylphosphonate ormalonylphosphonate, optionally wherein the phosphate analog is a4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy.Embodiment 39. The RNAi oligonucleotide of any one of the precedingembodiments, wherein at least one nucleotide of the oligonucleotide isconjugated to one or more targeting ligands.Embodiment 40. The RNAi oligonucleotide of embodiment 39, wherein eachtargeting ligand comprises a carbohydrate, amino sugar, cholesterol,polypeptide or lipid.Embodiment 41. The RNAi oligonucleotide of embodiment 39, wherein eachtargeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.Embodiment 42. The RNAi oligonucleotide of embodiment 35, wherein theGalNAc moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, atrivalent GalNAc moiety or a tetravalent GalNAc moiety.Embodiment 43. The RNAi oligonucleotide of any one of embodiments 16 to38, wherein up to 4 nucleotides of L of the stem-loop are eachconjugated to a monovalent GalNAc moiety.Embodiment 44. The RNAi oligonucleotide of any one of embodiments 1 to43, wherein the sense strand comprises a nucleotide sequence of any oneof SEQ ID NOs: 769-856 or 1519-1552.Embodiment 45. The RNAi oligonucleotide of any one of embodiments 1 to44, wherein the antisense strand comprises a nucleotide sequence of anyone of SEQ ID NOs: 857-944.Embodiment 46. The RNAi oligonucleotide of any one of embodiments 1 to45, wherein the sense strand and antisense strands comprise nucleotidesequences selected from the group consisting of:(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively.Embodiment 47. The RNAi oligonucleotide of any one of embodiments 1 to46, wherein the sense strand comprises a nucleotide sequence as setforth in SEQ ID NO: 786, wherein the antisense strand comprises anucleotide sequence as set forth in SEQ ID NO: 874.Embodiment 48. The RNAi oligonucleotide of any one of embodiments 1 to46, wherein the sense strand comprises a nucleotide sequence as setforth in SEQ ID NO: 787, wherein the antisense strand comprises anucleotide sequence as set forth in SEQ ID NO: 875.Embodiment 49. The RNAi oligonucleotide of any one of embodiments 1 to46, wherein the sense strand comprises a nucleotide sequence as setforth in SEQ ID NO: 1537, wherein the antisense strand comprises anucleotide sequence as set forth in SEQ ID NO: 929.Embodiment 50. The RNAi oligonucleotide of any one of embodiments 1 to46, wherein the sense strand comprises a nucleotide sequence as setforth in SEQ ID NO: 813, wherein the antisense strand comprises anucleotide sequence as set forth in SEQ ID NO: 901.Embodiment 51. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein all nucleotides comprising the sense strand and antisense strandare modified, wherein the antisense strand comprises a region ofcomplementarity to a NR1H3 mRNA target sequence of any one of SEQ IDNOs: 1-384, and wherein the region of complementarity is at least 15contiguous nucleotides in length.Embodiment 52. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein all nucleotides comprising the sense strand and antisense strandare modified, wherein the 4′-carbon of the sugar of the 5′-nucleotide ofthe antisense strand comprises a phosphate analog, wherein the antisensestrand comprises a region of complementarity to a NR1H3 mRNA targetsequence of any one of SEQ ID NOs: 1-384, and wherein the region ofcomplementarity is at least 15 contiguous nucleotides in length.Embodiment 53. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein all nucleotides comprising the sense strand and antisense strandare modified, wherein the 4′-carbon of the sugar of the 5′-nucleotide ofthe antisense strand comprises a phosphate analog, wherein the antisensestrand comprises a region of complementarity to a NR1H3 mRNA targetsequence of any one of SEQ ID NOs: 1-384, and wherein the region ofcomplementarity is at least 15 contiguous nucleotides in length.Embodiment 54. An RNAi oligonucleotide for reducing NR1H3 expression,the oligonucleotide comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a duplex region,wherein all nucleotides comprising the sense strand and the antisensestrand are modified, wherein the antisense strand and the sense strandcomprise one or more 2′-fluoro and 2′-O-methyl modified nucleotides andat least one phosphorothioate linkage, wherein the 4′-carbon of thesugar of the 5′-nucleotide of the antisense strand comprises a phosphateanalog, wherein the antisense strand comprises a region ofcomplementarity to a NR1H3 mRNA target sequence of any one of SEQ IDNOs: 1-384, and wherein the region of complementarity is at least 15contiguous nucleotides in length.Embodiment 55. The RNAi oligonucleotide of any one of embodiments 1-54,wherein the sense strand comprises a nucleotide sequence of any one ofSEQ ID NOs: 945-1032.Embodiment 56. The RNAi oligonucleotide of any one of embodiments 1-55,wherein the antisense strand comprises a nucleotide sequence of any oneof SEQ ID NOs: 1033-1120.Embodiment 57. The RNAi oligonucleotide of any one of embodiments 1-56,wherein the sense and antisense strands comprise nucleotide sequencesselected from the group consisting of:(a) SEQ ID NOs: 945 and 1033, respectively;(b) SEQ ID NOs: 946 and 1034, respectively;(c) SEQ ID NOs: 947 and 1035, respectively;(d) SEQ ID NOs: 948 and 1036, respectively;(e) SEQ ID NOs: 949 and 1037, respectively;(f) SEQ ID NOs: 950 and 1038, respectively;(g) SEQ ID NOs: 951 and 1039, respectively;(h) SEQ ID NOs: 952 and 1040, respectively;(i) SEQ ID NOs: 953 and 1041, respectively;(j) SEQ ID NOs: 954 and 1042, respectively;(k) SEQ ID NOs: 955 and 1043, respectively;(l) SEQ ID NOs: 956 and 1044 respectively;(m) SEQ ID NOs: 957 and 1045, respectively;(n) SEQ ID NOs: 958 and 1046, respectively;(o) SEQ ID NOs: 959 and 1047, respectively;(p) SEQ ID NOs: 960 and 1048, respectively;(q) SEQ ID NOs: 961 and 1049, respectively;(r) SEQ ID NOs: 962 and 1050, respectively;(s) SEQ ID NOs: 963 and 1051, respectively;(t) SEQ ID NOs: 964 and 1052, respectively;(u) SEQ ID NOs: 965 and 1053, respectively;(v) SEQ ID NOs: 966 and 1054, respectively;(w) SEQ ID NOs: 967 and 1055, respectively;(x) SEQ ID NOs: 968 and 1056, respectively;(y) SEQ ID NOs: 969 and 1057, respectively;(z) SEQ ID NOs: 970 and 1058, respectively;(aa) SEQ ID NOs: 971 and 1059, respectively;(bb) SEQ ID NOs: 972 and 1060, respectively;(cc) SEQ ID NOs: 973 and 1061, respectively;(dd) SEQ ID NOs: 974 and 1062, respectively;(ee) SEQ ID NOs: 975 and 1063, respectively;(ff) SEQ ID NOs: 976 and 1064, respectively;(gg) SEQ ID NOs: 977 and 1065, respectively;(hh) SEQ ID NOs: 978 and 1066, respectively;(ii) SEQ ID NOs: 979 and 1067, respectively;(jj) SEQ ID NOs: 980 and 1068, respectively;(kk) SEQ ID NOs: 981 and 1069, respectively;(ll) SEQ ID NOs: 982 and 1070, respectively;(mm) SEQ ID NOs: 983 and 1071, respectively;(nn) SEQ ID NOs: 984 and 1072, respectively;(oo) SEQ ID NOs: 985 and 1073, respectively;(pp) SEQ ID NOs: 986 and 1074, respectively;(qq) SEQ ID NOs: 987 and 1075, respectively;(rr) SEQ ID NOs: 988 and 1076, respectively;(ss) SEQ ID NOs: 989 and 1077, respectively;(tt) SEQ ID NOs: 990 and 1078, respectively;(uu) SEQ ID NOs: 991 and 1079, respectively;(vv) SEQ ID NOs: 992 and 1080, respectively;(ww) SEQ ID NOs: 993 and 1081, respectively;(xx) SEQ ID NOs: 994 and 1082, respectively;(yy) SEQ ID NOs: 995 and 1083, respectively;(zz) SEQ ID NOs: 996 and 1084, respectively;(aaa) SEQ ID NOs: 997 and 1085, respectively;(bbb) SEQ ID NOs: 998 and 1086, respectively;(ccc) SEQ ID NOs: 999 and 1087, respectively;(ddd) SEQ ID NOs: 1000 and 1088, respectively;(eee) SEQ ID NOs: 1001 and 1089, respectively;(fff) SEQ ID NOs: 1002 and 1090, respectively;(ggg) SEQ ID NOs: 1003 and 1091, respectively;(hhh) SEQ ID NOs: 1004 and 1092 respectively;(iii) SEQ ID NOs: 1005 and 1093 respectively;(jjj) SEQ ID NOs: 1006 and 1094, respectively;(kkk) SEQ ID NOs: 1007 and 1095, respectively;(lll) SEQ ID NOs: 1008 and 1096, respectively;(mmm) SEQ ID NOs: 1009 and 1097, respectively;(nnn) SEQ ID NOs: 1010 and 1098, respectively;(ooo) SEQ ID NOs: 1011 and 1099, respectively;(ppp) SEQ ID NOs: 1012 and 1100, respectively;(qqq) SEQ ID NOs: 1013 and 1101, respectively;(rrr) SEQ ID NOs: 1014 and 1102 respectively;(sss) SEQ ID NOs: 1015 and 1103, respectively;(ttt) SEQ ID NOs: 1016 and 1104, respectively;(uuu) SEQ ID NOs: 1017 and 1105, respectively;(vvv) SEQ ID NOs: 1018 and 1106, respectively;(www) SEQ ID NOs: 1019 and 1107, respectively;(xxx) SEQ ID NOs: 1020 and 1108, respectively;(yyy) SEQ ID NOs: 1021 and 1109, respectively;(zzz) SEQ ID NOs: 1022 and 1110, respectively;(aaaa) SEQ ID NOs: 1023 and 1111, respectively;(bbbb) SEQ ID NOs: 1024 and 1112, respectively;(cccc) SEQ ID NOs: 1025 and 1113, respectively;(dddd) SEQ ID NOs: 1026 and 1114, respectively;(eeee) SEQ ID NOs: 1027 and 1115, respectively;(ffff) SEQ ID NOs: 1028 and 1116, respectively;(gggg) SEQ ID NOs: 1029 and 1117, respectively;(hhhh) SEQ ID NOs: 1030 and 1118, respectively;(iiii) SEQ ID NOs: 1031 and 1119, respectively; and,(jjjj) SEQ ID NOs: 1032 and 1120, respectively.Embodiment 58. The RNAi oligonucleotide of any one of embodiments 1-57,wherein the sense and antisense strands comprise the nucleotidesequences set forth in SEQ ID NOs: 963 and 1051, respectively.Embodiment 59. The RNAi oligonucleotide of any one of embodiments 1-57,wherein the sense and antisense strands comprise the nucleotidesequences set forth in SEQ ID NOs: 964 and 1052, respectively.Embodiment 60. The RNAi oligonucleotide of any one of embodiments 1-57,wherein the sense and antisense strands comprise the nucleotidesequences set forth in SEQ ID NOs: 1006 and 1094, respectively.Embodiment 61. The RNAi oligonucleotide of any one of embodiments 1-57,wherein the sense and antisense strands comprise the nucleotidesequences set forth in SEQ ID NOs: 1018 and 1106, respectively.Embodiment 62. An RNAi oligonucleotide for inhibiting expression ofNR1H3, wherein said dsRNA comprises a sense strand and an antisensestrand, the antisense strand comprising a region of complementarity to aNR1H3 RNA transcript, wherein the sense strand comprises the sequenceand all of the modifications of5′-mCs-mU-mC-mA-mA-mG-mG-fA-fU-fU-fU-mC-mA-mG-mU-mU-mA-mU-mA-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 963), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-40-mUs]-fUs-fAs-fU-fA-mA-fC-mU-mG-fA-mA-mA-mU-fC-mC-mU-mU-mG-mA-mGs-mGs-mG-3′(SEQ ID NO: 1051), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=

Embodiment 63. An RNAi oligonucleotide for inhibiting expression ofNR1H3, wherein said dsRNA comprises a sense strand and an antisensestrand, the antisense strand comprising a region of complementarity to aNR1H3 RNA transcript, wherein the sense strand comprises the sequenceand all of the modifications of5′-mUs-mC-mA-mA-mG-mG-mA-fU-fU-fU-fC-mA-mG-mU-mU-mA-mU-mA-mA-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 964) and wherein the antisense strand comprises the sequenceand all of the modifications of5′-[MePhosphonate-4O-mUs]-fUs-fUs-fA-fU-mA-fA-mC-mU-fG-mA-mA-mA-fU-mC-mC-mU-mU-mG-mAs-mGs-mG-3′(SEQ ID NO: 1052), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=

Embodiment 64. An RNAi oligonucleotide for inhibiting expression ofNR1H3, wherein said dsRNA comprises a sense strand and an antisensestrand, the antisense strand comprising a region of complementarity to aNR1H3 RNA transcript, wherein the sense strand comprises the sequenceand all of the modifications of5′-mAs-mG-mC-mA-mG-mC-mG-fU-fC-fC-fA-mC-mU-mC-mA-mG-mA-mG-mC-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 1006), and wherein the antisense strand comprises thesequence and all of the modifications of5′[MePhosphonate-4O-mUs]-fGs-fCs-fU-fC-mU-fG-mA-mG-fU-mG-mG-mA-fC-mG-mC-mU-mG-mC-mUs-mGs-mG-3′(SEQ ID NO: 1094), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fU=2′-F ribonucleosides; s=phosphorothioate, and whereinademA-GalNAc=

Embodiment 65. A double stranded RNAi oligonucleotide (dsRNAi) forinhibiting expression of NR1H3, wherein said dsRNA comprises a sensestrand and an antisense strand, the antisense strand comprising a regionof complementarity to a NR1H3 RNA transcript, wherein the sense strandcomprises the sequence and all of the modifications of5′-mAs-mU-mG-mU-mG-mC-mA-fC-fG-fA-fA-mU-mG-mA-mC-mU-mG-mU-mU-mA-mG-mC-mA-mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC-3′(SEQ ID NO: 1018), and wherein the antisense strand comprises thesequence and all of the modifications of5′-[MePhosphonate-4O-mUs]-fAs-fAs-fC-fA-mG-fU-mC-mA-fU-mU-mC-mG-fU-mG-mC-mA-mC-mA-mUs-mGs-mG-3′(SEQ ID NO: 1106), wherein mC, mA, mG, and mU=2′-OMe ribonucleosides;fA, fC, fG, and fUJ=2′-F ribonucleosides; s=phosphorothioate, andwherein ademA-GalNAc=

Embodiment 66. A method for treating a subject having a disease,disorder or condition associated with NR1H3 expression, the methodcomprising administering to the subject a therapeutically effectiveamount of the RNAi oligonucleotide of any one of the precedingembodiments, or pharmaceutical composition thereof, thereby treating thesubject.Embodiment 67. A pharmaceutical composition comprising the RNAioligonucleotide of any one of embodiments 1 to 65, and apharmaceutically acceptable carrier, delivery agent or excipient.Embodiment 68. A method of delivering an oligonucleotide to a subject,the method comprising administering pharmaceutical composition ofembodiment 67 to the subject.Embodiment 69. A method for reducing NR1H3 expression in a cell, apopulation of cells or a subject, the method comprising the step of:

i. contacting the cell or the population of cells with the RNAioligonucleotide of any one of embodiments 1 to 65, or the pharmaceuticalcomposition of embodiment 67; or

ii. administering to the subject the RNAi oligonucleotide of any one ofembodiments 1 to 65, or the pharmaceutical composition of embodiment 67.

Embodiment 70. The method of embodiment 69, wherein reducing NR1H3expression comprises reducing an amount or level of NR1H3 mRNA, anamount or level of NR1H3 protein, or both.Embodiment 71. The method of embodiment 69 or 70, wherein the subjecthas a disease, disorder or condition associated with NR1H3 expression.Embodiment 72. The method of embodiment 66 or 71, wherein the disease,disorder or condition associated with NR1H3 expression is non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),multiple sclerosis, or systemic lupus erythematosus.Embodiment 73. The method of any one of embodiments 66 and 69 to 72,wherein the RNAi oligonucleotide, or pharmaceutical composition, isadministered in combination with a second composition or therapeuticagent.Embodiment 74. A method for treating a subject having a disease,disorder or condition associated with NR1H3 expression, the methodcomprising administering to the subject a therapeutically effectiveamount of an RNAi oligonucleotide comprising a sense strand and anantisense strand, wherein the sense strand and the antisense strand forma duplex region, wherein the antisense strand comprises a region ofcomplementarity to a NR1H3 mRNA target sequence of any one of SEQ IDNOs: 1-384, and wherein the region of complementarity is at least 15contiguous nucleotides in length.Embodiment 75. A method for treating a subject having a disease,disorder or condition associated with NR1H3 expression, the methodcomprising administering to the subject a therapeutically effectiveamount of an RNAi oligonucleotide comprising a sense strand and anantisense strand selected from a row set forth in Table 4 or Table 5, orpharmaceutical composition thereof, thereby treating the subject.Embodiment 76. A method for treating a subject having a disease,disorder or condition associated with NR1H3 expression, the methodcomprising administering to the subject a therapeutically effectiveamount of an RNAi oligonucleotide comprising a sense strand and anantisense strand, wherein the sense strand and antisense strandscomprise nucleotide sequences selected from the group consisting of:(a) SEQ ID NOs: 769 and 857, respectively;(b) SEQ ID NOs: 770 and 858, respectively;(c) SEQ ID NOs: 771 and 859, respectively;(d) SEQ ID NOs: 772 and 860, respectively;(e) SEQ ID NOs: 773 and 861, respectively;(f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively;(h) SEQ ID NOs: 776 and 864, respectively;(i) SEQ ID NOs: 777 and 865, respectively;(j) SEQ ID NOs: 778 and 866, respectively;(k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively;(m) SEQ ID NOs: 781 and 869, respectively;(n) SEQ ID NOs: 782 and 870, respectively;(o) SEQ ID NOs: 783 and 871, respectively;(p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively;(r) SEQ ID NOs: 786 and 874, respectively;(s) SEQ ID NOs: 787 and 875, respectively;(t) SEQ ID NOs: 788 and 876, respectively;(u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively;(w) SEQ ID NOs: 791 and 879, respectively;(x) SEQ ID NOs: 792 and 880, respectively;(y) SEQ ID NOs: 793 and 881, respectively;(z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively;(bb) SEQ ID NOs: 796 and 884, respectively;(cc) SEQ ID NOs: 797 and 885, respectively;(dd) SEQ ID NOs: 798 and 886, respectively;(ee) SEQ ID NOs: 799 and 887, respectively;(ff) SEQ ID NOs: 800 and 888, respectively;(gg) SEQ ID NOs: 801 and 889, respectively;(hh) SEQ ID NOs: 802 and 890, respectively;(ii) SEQ ID NOs: 803 and 891, respectively;(jj) SEQ ID NOs: 804 and 892, respectively;(kk) SEQ ID NOs: 805 and 893, respectively;(ll) SEQ ID NOs: 806 and 894, respectively;(mm) SEQ ID NOs: 807 and 895, respectively;(nn) SEQ ID NOs: 808 and 896, respectively;(oo) SEQ ID NOs: 809 and 897, respectively;(pp) SEQ ID NOs: 810 and 898, respectively;(qq) SEQ ID NOs: 811 and 899, respectively;(rr) SEQ ID NOs: 812 and 900, respectively;(ss) SEQ ID NOs: 813 and 901, respectively;(tt) SEQ ID NOs: 814 and 902, respectively;(uu) SEQ ID NOs: 815 and 903, respectively;(vv) SEQ ID NOs: 816 and 904, respectively;(ww) SEQ ID NOs: 817 and 905, respectively;(xx) SEQ ID NOs: 818 and 906, respectively;(yy) SEQ ID NOs: 819 and 907, respectively;(zz) SEQ ID NOs: 820 and 908, respectively;(aaa) SEQ ID NOs: 821 and 909, respectively;(bbb) SEQ ID NOs: 822 and 910, respectively;(ccc) SEQ ID NOs: 823 and 911, respectively;(ddd) SEQ ID NOs: 824 and 912, respectively;(eee) SEQ ID NOs: 825 and 913, respectively;(fff) SEQ ID NOs: 826 and 914, respectively;(ggg) SEQ ID NOs: 827 and 915, respectively;(hhh) SEQ ID NOs: 828 and 916, respectively;(iii) SEQ ID NOs: 829 and 917, respectively;(jjj) SEQ ID NOs: 830 and 918, respectively;(kkk) SEQ ID NOs: 831 and 919, respectively;(lll) SEQ ID NOs: 832 and 920, respectively;(mmm) SEQ ID NOs: 833 and 921, respectively;(nnn) SEQ ID NOs: 834 and 922, respectively;(ooo) SEQ ID NOs: 835 and 923, respectively;(ppp) SEQ ID NOs: 836 and 924, respectively;(qqq) SEQ ID NOs: 837 and 925, respectively;(rrr) SEQ ID NOs: 838 and 926, respectively;(sss) SEQ ID NOs: 839 and 927, respectively;(ttt) SEQ ID NOs: 840 and 928, respectively;(uuu) SEQ ID NOs: 1537 and 929, respectively;(vvv) SEQ ID NOs: 842 and 930, respectively;(www) SEQ ID NOs: 843 and 931, respectively;(xxx) SEQ ID NOs: 844 and 932, respectively;(yyy) SEQ ID NOs: 845 and 933, respectively;(zzz) SEQ ID NOs: 846 and 934, respectively;(aaaa) SEQ ID NOs: 847 and 935, respectively;(bbbb) SEQ ID NOs: 848 and 936, respectively;(cccc) SEQ ID NOs: 849 and 937, respectively;(dddd) SEQ ID NOs: 850 and 938, respectively;(eeee) SEQ ID NOs: 851 and 939, respectively;(ffff) SEQ ID NOs: 852 and 940, respectively;(gggg) SEQ ID NOs: 853 and 941, respectively;(hhhh) SEQ ID NOs: 854 and 942, respectively;(iiii) SEQ ID NOs: 855 and 943, respectively; and,(jjjj) SEQ ID NOs: 856 and 944, respectively.Embodiment 77. The method of embodiment 76, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 786, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 874.Embodiment 78. The method of embodiment 76, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 787, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 875.Embodiment 79. The method of embodiment 76, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 1537, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 929.Embodiment 80. The method of embodiment 76, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 813, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 901.Embodiment 81. A method for treating a subject having a disease,disorder or condition associated with NR1H3 expression, the methodcomprising administering to the subject a therapeutically effectiveamount of an RNAi oligonucleotide comprising a sense strand and anantisense strand, wherein the sense strand and antisense strandscomprise nucleotide sequences selected from the group consisting of:(a) SEQ ID NOs: 945 and 1033, respectively;(b) SEQ ID NOs: 946 and 1034, respectively;(c) SEQ ID NOs: 947 and 1035, respectively;(d) SEQ ID NOs: 948 and 1036, respectively;(e) SEQ ID NOs: 949 and 1037, respectively;(f) SEQ ID NOs: 950 and 1038, respectively;(g) SEQ ID NOs: 951 and 1039, respectively;(h) SEQ ID NOs: 952 and 1040, respectively;(i) SEQ ID NOs: 953 and 1041, respectively;(j) SEQ ID NOs: 954 and 1042, respectively;(k) SEQ ID NOs: 955 and 1043, respectively;(l) SEQ ID NOs: 956 and 1044 respectively;(m) SEQ ID NOs: 957 and 1045, respectively;(n) SEQ ID NOs: 958 and 1046, respectively;(o) SEQ ID NOs: 959 and 1047, respectively;(p) SEQ ID NOs: 960 and 1048, respectively;(q) SEQ ID NOs: 961 and 1049, respectively;(r) SEQ ID NOs: 962 and 1050, respectively;(s) SEQ ID NOs: 963 and 1051, respectively;(t) SEQ ID NOs: 964 and 1052, respectively;(u) SEQ ID NOs: 965 and 1053, respectively;(v) SEQ ID NOs: 966 and 1054, respectively;(w) SEQ ID NOs: 967 and 1055, respectively;(x) SEQ ID NOs: 968 and 1056, respectively;(y) SEQ ID NOs: 969 and 1057, respectively;(z) SEQ ID NOs: 970 and 1058, respectively;(aa) SEQ ID NOs: 971 and 1059, respectively;(bb) SEQ ID NOs: 972 and 1060, respectively;(cc) SEQ ID NOs: 973 and 1061, respectively;(dd) SEQ ID NOs: 974 and 1062, respectively;(ee) SEQ ID NOs: 975 and 1063, respectively;(ff) SEQ ID NOs: 976 and 1064, respectively;(gg) SEQ ID NOs: 977 and 1065, respectively;(hh) SEQ ID NOs: 978 and 1066, respectively;(ii) SEQ ID NOs: 979 and 1067, respectively;(jj) SEQ ID NOs: 980 and 1068, respectively;(kk) SEQ ID NOs: 981 and 1069, respectively;(ll) SEQ ID NOs: 982 and 1070, respectively;(mm) SEQ ID NOs: 983 and 1071, respectively;(nn) SEQ ID NOs: 984 and 1072, respectively;(oo) SEQ ID NOs: 985 and 1073, respectively;(pp) SEQ ID NOs: 986 and 1074, respectively;(qq) SEQ ID NOs: 987 and 1075, respectively;(rr) SEQ ID NOs: 988 and 1076, respectively;(ss) SEQ ID NOs: 989 and 1077, respectively;(tt) SEQ ID NOs: 990 and 1078, respectively;(uu) SEQ ID NOs: 991 and 1079, respectively;(vv) SEQ ID NOs: 992 and 1080, respectively;(ww) SEQ ID NOs: 993 and 1081, respectively;(xx) SEQ ID NOs: 994 and 1082, respectively;(yy) SEQ ID NOs: 995 and 1083, respectively;(zz) SEQ ID NOs: 996 and 1084, respectively;(aaa) SEQ ID NOs: 997 and 1085, respectively;(bbb) SEQ ID NOs: 998 and 1086, respectively;(ccc) SEQ ID NOs: 999 and 1087, respectively;(ddd) SEQ ID NOs: 1000 and 1088, respectively;(eee) SEQ ID NOs: 1001 and 1089, respectively;(fff) SEQ ID NOs: 1002 and 1090, respectively;(ggg) SEQ ID NOs: 1003 and 1091, respectively;(hhh) SEQ ID NOs: 1004 and 1092 respectively;(iii) SEQ ID NOs: 1005 and 1093 respectively;(jjj) SEQ ID NOs: 1006 and 1094, respectively;(kkk) SEQ ID NOs: 1007 and 1095, respectively;(lll) SEQ ID NOs: 1008 and 1096, respectively;(mmm) SEQ ID NOs: 1009 and 1097, respectively;(nnn) SEQ ID NOs: 1010 and 1098, respectively;(ooo) SEQ ID NOs: 1011 and 1099, respectively;(ppp) SEQ ID NOs: 1012 and 1100, respectively;(qqq) SEQ ID NOs: 1013 and 1101, respectively;(rrr) SEQ ID NOs: 1014 and 1102 respectively;(sss) SEQ ID NOs: 1015 and 1103, respectively;(ttt) SEQ ID NOs: 1016 and 1104, respectively;(uuu) SEQ ID NOs: 1017 and 1105, respectively;(vvv) SEQ ID NOs: 1018 and 1106, respectively;(www) SEQ ID NOs: 1019 and 1107, respectively;(xxx) SEQ ID NOs: 1020 and 1108, respectively;(yyy) SEQ ID NOs: 1021 and 1109, respectively;(zzz) SEQ ID NOs: 1022 and 1110, respectively;(aaaa) SEQ ID NOs: 1023 and 1111, respectively;(bbbb) SEQ ID NOs: 1024 and 1112, respectively;(cccc) SEQ ID NOs: 1025 and 1113, respectively;(dddd) SEQ ID NOs: 1026 and 1114, respectively;(eeee) SEQ ID NOs: 1027 and 1115, respectively;(ffff) SEQ ID NOs: 1028 and 1116, respectively;(gggg) SEQ ID NOs: 1029 and 1117, respectively;(hhhh) SEQ ID NOs: 1030 and 1118, respectively;(iiii) SEQ ID NOs: 1031 and 1119, respectively, and;(jjjj) SEQ ID NOs: 1032 and 1120, respectively.Embodiment 82. The method of embodiment 81, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 963, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 1051.Embodiment 83. The method of embodiment 81, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 964, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 1052.Embodiment 84. The method of embodiment 81, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 1006, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 1094.Embodiment 85. The method of embodiment 81, wherein the sense strandcomprises a nucleotide sequence as set forth in SEQ ID NO: 1018, whereinthe antisense strand comprises a nucleotide sequence as set forth in SEQID NO: 1106.Embodiment 86. The method of any one of embodiments 74 to 85, whereinthe disease, disorder or condition associated with NR1H3 expression isnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), multiple sclerosis, or systemic lupus erythematosus.Embodiment 87. Use of the RNAi oligonucleotide of any one of embodiments1 to 65, or the pharmaceutical composition of embodiment 67, in themanufacture of a medicament for the treatment of a disease, disorder orcondition associated with NR1H3 expression, optionally for the treatmentof non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), multiple sclerosis, or systemic lupuserythematosus.Embodiment 88. The RNAi oligonucleotide of any one of embodiments 1 to65, or the pharmaceutical composition of embodiment 67, for use, oradaptable for use, in the treatment of a disease, disorder or conditionassociated with NR1H3 expression, optionally for the treatment ofnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), multiple sclerosis, or systemic lupus erythematosus.Embodiment 89. A kit comprising the RNAi oligonucleotide of any one ofembodiments 1 to 65, an optional pharmaceutically acceptable carrier,and a package insert comprising instructions for administration to asubject having a disease, disorder or condition associated with NR1H3expression.Embodiment 90. The use of embodiment 87, the RNAi oligonucleotide orpharmaceutical composition for use, or adaptable for use, of embodiment88, or the kit of embodiment 89, wherein the disease, disorder orcondition associated with NR1H3 expression is non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), multiplesclerosis, or systemic lupus erythematosus.Embodiment 91. A double stranded RNAi oligonucleotide (dsRNAi) forreducing NR1H3 expression, the oligonucleotide comprising a sense strandand an antisense strand, wherein the sense strand and the antisensestrand form a duplex region, wherein

(i) the sense strand comprises a nucleotide sequence comprising at least15, 17, or 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, ofa portion of a nucleotide sequence of any one of SEQ ID NOs: 1-384, SEQID NOs: 1125-1511, SEQ ID NOs: 769-856, SEQ ID NOs: 1519-1552 or SEQ IDNOs: 945-1032; and

(ii) the antisense strand comprises a nucleotide sequence comprising atleast 15, 17, or 19 contiguous nucleotides, with 0, 1, 2, or 3mismatches, of a portion of a nucleotide sequence of any one of SEQ IDNOs: 385-768, SEQ ID NOs 857-944, SEQ ID NOs 1512-1515, or SEQ ID NOs:1033-1120.

1. A double stranded RNAi oligonucleotide (dsRNAi) for reducing NR1H3expression, the oligonucleotide comprising a sense strand and anantisense strand, wherein the sense strand and the antisense strand forma duplex region, wherein the sense and antisense strands comprisenucleotide sequences selected from the group consisting of: (a) SEQ IDNOs: 945 and 1033, respectively; (b) SEQ ID NOs: 946 and 1034,respectively; (c) SEQ ID NOs: 947 and 1035, respectively; (d) SEQ IDNOs: 948 and 1036, respectively; (e) SEQ ID NOs: 949 and 1037,respectively; (f) SEQ ID NOs: 950 and 1038, respectively; (g) SEQ IDNOs: 951 and 1039, respectively; (h) SEQ ID NOs: 952 and 1040,respectively; (i) SEQ ID NOs: 953 and 1041, respectively; (j) SEQ IDNOs: 954 and 1042, respectively; (k) SEQ ID NOs: 955 and 1043,respectively; (l) SEQ ID NOs: 956 and 1044 respectively; (m) SEQ ID NOs:957 and 1045, respectively; (n) SEQ ID NOs: 958 and 1046, respectively;(o) SEQ ID NOs: 959 and 1047, respectively; (p) SEQ ID NOs: 960 and1048, respectively; (q) SEQ ID NOs: 961 and 1049, respectively; (r) SEQID NOs: 962 and 1050, respectively; (s) SEQ ID NOs: 963 and 1051,respectively; (t) SEQ ID NOs: 964 and 1052, respectively; (u) SEQ IDNOs: 965 and 1053, respectively; (v) SEQ ID NOs: 966 and 1054,respectively; (w) SEQ ID NOs: 967 and 1055, respectively; (x) SEQ IDNOs: 968 and 1056, respectively; (y) SEQ ID NOs: 969 and 1057,respectively; (z) SEQ ID NOs: 970 and 1058, respectively; (aa) SEQ IDNOs: 971 and 1059, respectively; (bb) SEQ ID NOs: 972 and 1060,respectively; (cc) SEQ ID NOs: 973 and 1061, respectively; (dd) SEQ IDNOs: 974 and 1062, respectively; (ee) SEQ ID NOs: 975 and 1063,respectively; (ff) SEQ ID NOs: 976 and 1064, respectively; (gg) SEQ IDNOs: 977 and 1065, respectively; (hh) SEQ ID NOs: 978 and 1066,respectively; (ii) SEQ ID NOs: 979 and 1067, respectively; (jj) SEQ IDNOs: 980 and 1068, respectively; (kk) SEQ ID NOs: 981 and 1069,respectively; (ll) SEQ ID NOs: 982 and 1070, respectively; (mm) SEQ IDNOs: 983 and 1071, respectively; (nn) SEQ ID NOs: 984 and 1072,respectively; (oo) SEQ ID NOs: 985 and 1073, respectively; (pp) SEQ IDNOs: 986 and 1074, respectively; (qq) SEQ ID NOs: 987 and 1075,respectively; (rr) SEQ ID NOs: 988 and 1076, respectively; (ss) SEQ IDNOs: 989 and 1077, respectively; (tt) SEQ ID NOs: 990 and 1078,respectively; (uu) SEQ ID NOs: 991 and 1079, respectively; (vv) SEQ IDNOs: 992 and 1080, respectively; (ww) SEQ ID NOs: 993 and 1081,respectively; (xx) SEQ ID NOs: 994 and 1082, respectively; (yy) SEQ IDNOs: 995 and 1083, respectively; (zz) SEQ ID NOs: 996 and 1084,respectively; (aaa) SEQ ID NOs: 997 and 1085, respectively; (bbb) SEQ IDNOs: 998 and 1086, respectively; (ccc) SEQ ID NOs: 999 and 1087,respectively; (ddd) SEQ ID NOs: 1000 and 1088, respectively; (eee) SEQID NOs: 1001 and 1089, respectively; (fff) SEQ ID NOs: 1002 and 1090,respectively; (ggg) SEQ ID NOs: 1003 and 1091, respectively; (hhh) SEQID NOs: 1004 and 1092 respectively; (iii) SEQ ID NOs: 1005 and 1093respectively; (jjj) SEQ ID NOs: 1006 and 1094, respectively; (kkk) SEQID NOs: 1007 and 1095, respectively; (lll) SEQ ID NOs: 1008 and 1096,respectively; (mmm) SEQ ID NOs: 1009 and 1097, respectively; (nnn) SEQID NOs: 1010 and 1098, respectively; (ooo) SEQ ID NOs: 1011 and 1099,respectively; (ppp) SEQ ID NOs: 1012 and 1100, respectively; (qqq) SEQID NOs: 1013 and 1101, respectively; (rrr) SEQ ID NOs: 1014 and 1102respectively; (sss) SEQ ID NOs: 1015 and 1103, respectively; (ttt) SEQID NOs: 1016 and 1104, respectively; (uuu) SEQ ID NOs: 1017 and 1105,respectively; (vvv) SEQ ID NOs: 1018 and 1106, respectively; (www) SEQID NOs: 1019 and 1107, respectively; (xxx) SEQ ID NOs: 1020 and 1108,respectively; (yyy) SEQ ID NOs: 1021 and 1109, respectively; (zzz) SEQID NOs: 1022 and 1110, respectively; (aaaa) SEQ ID NOs: 1023 and 1111,respectively; (bbbb) SEQ ID NOs: 1024 and 1112, respectively; (cccc) SEQID NOs: 1025 and 1113, respectively; (dddd) SEQ ID NOs: 1026 and 1114,respectively; (eeee) SEQ ID NOs: 1027 and 1115, respectively; (ffff) SEQID NOs: 1028 and 1116, respectively; (gggg) SEQ ID NOs: 1029 and 1117,respectively; (hhhh) SEQ ID NOs: 1030 and 1118, respectively; (iiii) SEQID NOs: 1031 and 1119, respectively; and, (jjjj) SEQ ID NOs: 1032 and1120, respectively.
 2. The double stranded RNAi oligonucleotide (dsRNAi)of claim 1, wherein the sense and antisense strands comprise thenucleotide sequences set forth in SEQ ID NOs: 964 and 1052,respectively.
 3. The double stranded RNAi oligonucleotide (dsRNAi) ofclaim 1, wherein the sense and antisense strands comprise the nucleotidesequences set forth in SEQ ID NOs: 963 and 1051, respectively.
 4. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 1, wherein thesense and antisense strands comprise the nucleotide sequences set forthin SEQ ID NOs: 1006 and 1094, respectively.
 5. The double stranded RNAioligonucleotide (dsRNAi) of claim 1, wherein the sense and antisensestrands comprise the nucleotide sequences set forth in SEQ ID NOs: 1018and 1106, respectively.
 6. A double stranded RNAi oligonucleotide(dsRNAi) for reducing NR1H3 expression, the oligonucleotide comprising asense strand and an antisense strand, wherein the sense strand and theantisense strand form a duplex region, wherein (i) the sense strandcomprises a nucleotide sequence comprising at least 15, 17, or 19contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of anucleotide sequence of any one of SEQ ID NOs: 1-384, or SEQ ID NOs769-856, or SEQ ID NOs 1519-1552; and (ii) the antisense strandcomprises a nucleotide sequence comprising at least 15, 17, or 19contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of anucleotide sequence of any one of SEQ ID NOs: 385-768, SEQ ID NOs857-944, or SEQ ID NOs 1512-1515.
 7. The double stranded RNAioligonucleotide (dsRNAi) of claim 6, wherein the sense strand andantisense strands comprise nucleotide sequences selected from the groupconsisting of: (a) SEQ ID NOs: 769 and 857, respectively; (b) SEQ IDNOs: 770 and 858, respectively; (c) SEQ ID NOs: 771 and 859,respectively; (d) SEQ ID NOs: 772 and 860, respectively; (e) SEQ ID NOs:773 and 861, respectively; (f) SEQ ID NOs: 774 and 862, respectively;(g) SEQ ID NOs: 775 and 863, respectively; (h) SEQ ID NOs: 776 and 864,respectively; (i) SEQ ID NOs: 777 and 865, respectively; (j) SEQ ID NOs:778 and 866, respectively; (k) SEQ ID NOs: 779 and 867, respectively;(l) SEQ ID NOs: 780 and 868, respectively; (m) SEQ ID NOs: 781 and 869,respectively; (n) SEQ ID NOs: 782 and 870, respectively; (o) SEQ ID NOs:783 and 871, respectively; (p) SEQ ID NOs: 784 and 872, respectively;(q) SEQ ID NOs: 785 and 873, respectively; (r) SEQ ID NOs: 786 and 874,respectively; (s) SEQ ID NOs: 787 and 875, respectively; (t) SEQ ID NOs:788 and 876, respectively; (u) SEQ ID NOs: 789 and 877, respectively;(v) SEQ ID NOs: 790 and 878, respectively; (w) SEQ ID NOs: 791 and 879,respectively; (x) SEQ ID NOs: 792 and 880, respectively; (y) SEQ ID NOs:793 and 881, respectively; (z) SEQ ID NOs: 794 and 882, respectively;(aa) SEQ ID NOs: 795 and 883, respectively; (bb) SEQ ID NOs: 796 and884, respectively; (cc) SEQ ID NOs: 797 and 885, respectively; (dd) SEQID NOs: 798 and 886, respectively; (ee) SEQ ID NOs: 799 and 887,respectively; (ff) SEQ ID NOs: 800 and 888, respectively; (gg) SEQ IDNOs: 801 and 889, respectively; (hh) SEQ ID NOs: 802 and 890,respectively; (ii) SEQ ID NOs: 803 and 891, respectively; (jj) SEQ IDNOs: 804 and 892, respectively; (kk) SEQ ID NOs: 805 and 893,respectively; (ll) SEQ ID NOs: 806 and 894, respectively; (mm) SEQ IDNOs: 807 and 895, respectively; (nn) SEQ ID NOs: 808 and 896,respectively; (oo) SEQ ID NOs: 809 and 897, respectively; (pp) SEQ IDNOs: 810 and 898, respectively; (qq) SEQ ID NOs: 811 and 899,respectively; (rr) SEQ ID NOs: 812 and 900, respectively; (ss) SEQ IDNOs: 813 and 901, respectively; (tt) SEQ ID NOs: 814 and 902,respectively; (uu) SEQ ID NOs: 815 and 903, respectively; (vv) SEQ IDNOs: 816 and 904, respectively; (ww) SEQ ID NOs: 817 and 905,respectively; (xx) SEQ ID NOs: 818 and 906, respectively; (yy) SEQ IDNOs: 819 and 907, respectively; (zz) SEQ ID NOs: 820 and 908,respectively; (aaa) SEQ ID NOs: 821 and 909, respectively; (bbb) SEQ IDNOs: 822 and 910, respectively; (ccc) SEQ ID NOs: 823 and 911,respectively; (ddd) SEQ ID NOs: 824 and 912, respectively; (eee) SEQ IDNOs: 825 and 913, respectively; (fff) SEQ ID NOs: 826 and 914,respectively; (ggg) SEQ ID NOs: 827 and 915, respectively; (hhh) SEQ IDNOs: 828 and 916, respectively; (iii) SEQ ID NOs: 829 and 917,respectively; (jjj) SEQ ID NOs: 830 and 918, respectively; (kkk) SEQ IDNOs: 831 and 919, respectively; (lll) SEQ ID NOs: 832 and 920,respectively; (mmm) SEQ ID NOs: 833 and 921, respectively; (nnn) SEQ IDNOs: 834 and 922, respectively; (ooo) SEQ ID NOs: 835 and 923,respectively; (ppp) SEQ ID NOs: 836 and 924, respectively; (qqq) SEQ IDNOs: 837 and 925, respectively; (rrr) SEQ ID NOs: 838 and 926,respectively; (sss) SEQ ID NOs: 839 and 927, respectively; (ttt) SEQ IDNOs: 840 and 928, respectively; (uuu) SEQ ID NOs: 1537 and 929,respectively; (vvv) SEQ ID NOs: 842 and 930, respectively; (www) SEQ IDNOs: 843 and 931, respectively; (xxx) SEQ ID NOs: 844 and 932,respectively; (yyy) SEQ ID NOs: 845 and 933, respectively; (zzz) SEQ IDNOs: 846 and 934, respectively; (aaaa) SEQ ID NOs: 847 and 935,respectively; (bbbb) SEQ ID NOs: 848 and 936, respectively; (cccc) SEQID NOs: 849 and 937, respectively; (dddd) SEQ ID NOs: 850 and 938,respectively; (eeee) SEQ ID NOs: 851 and 939, respectively; (ffff) SEQID NOs: 852 and 940, respectively; (gggg) SEQ ID NOs: 853 and 941,respectively; (hhhh) SEQ ID NOs: 854 and 942, respectively; (iiii) SEQID NOs: 855 and 943, respectively; and, (jjjj) SEQ ID NOs: 856 and 944,respectively.
 8. An RNAi oligonucleotide for reducing growth (NR1H3)expression, the oligonucleotide comprising a sense strand and anantisense strand, wherein the sense strand and the antisense strand forma duplex region, wherein the antisense strand comprises a region ofcomplementarity to a NR1H3 mRNA target sequence of any one of SEQ IDNOs: 1125-1511 and wherein the region of complementarity is at least 15contiguous nucleotides in length.
 9. The double stranded RNAioligonucleotide (dsRNAi) of claim 6, wherein the 3′ end of the sensestrand comprises a stem-loop set forth as S1-L-S2, and wherein (i) S1 iscomplementary to S2, and S1 and S2 have the same length and are each1-10 nucleotides in length; and (ii) L forms a loop between S1 and S2 of3-5 nucleotides in length.
 10. The double stranded RNAi oligonucleotide(dsRNAi) of claim 9, wherein S1 and S2 are 6 nucleotides in length; andL is a triloop or a tetraloop.
 11. The double stranded RNAioligonucleotide (dsRNAi) of claim 10, wherein L is a tetraloopcomprising the sequence 5′-GAAA-3′.
 12. The double stranded RNAioligonucleotide (dsRNAi) of claim 9, wherein the stem-loop comprises thesequence 5′-GCAGCCGAAAGGCUGC-3′ (SEQ ID NO: 1121).
 13. The doublestranded RNAi oligonucleotide (dsRNAi) of claim 9, wherein at least onenucleotide of the oligonucleotide is conjugated to one or more targetingligands.
 14. The double stranded RNAi oligonucleotide (dsRNAi) of claim13, wherein each targeting ligand comprises a carbohydrate, amino sugar,cholesterol, polypeptide or lipid.
 15. The double stranded RNAioligonucleotide (dsRNAi) of claim 13, wherein the targeting ligand is ahepatocyte targeting ligand and each targeting ligand comprises aN-acetylgalactosamine (GalNAc) moiety.
 16. The double stranded RNAioligonucleotide (dsRNAi) of claim 15, wherein the N-acetylgalactosamine(GalNAc) moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety,a trivalent GalNAc moiety or a tetravalent GalNAc moiety.
 17. The doublestranded RNAi oligonucleotide (dsRNAi) of claim 13, wherein the stemloop comprises one or more targeting ligands conjugated to one or morenucleotides of the stem loop or the loop comprises one or more targetingligands conjugated to one or more nucleotides of the loop.
 18. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 17, wherein theone or more targeting ligands is conjugated to one or more nucleotidesof the loop, wherein the loop comprises 4 nucleotides numbered 1-4 from5′ to 3′, wherein nucleotides at positions 2, 3, and 4 each comprise oneor more targeting ligands, and wherein the targeting ligands are thesame or different.
 19. The double stranded RNAi oligonucleotide (dsRNAi)of claim 17, wherein the targeting ligand is a hepatocyte targetingligand and each targeting ligand comprises a N-acetylgalactosamine(GalNAc) moiety.
 20. The double stranded RNAi oligonucleotide (dsRNAi)of claim 19, wherein the N-acetylgalactosamine (GalNAc) moiety is amonovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAcmoiety or a tetravalent GalNAc moiety.
 21. The double stranded RNAioligonucleotide (dsRNAi) of claim 17, wherein the targeting ligand is ahepatocyte targeting ligand and up to 4 nucleotides of L of thestem-loop are each conjugated to a monovalent GalNAc moiety.
 22. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 9, comprising atleast one modified nucleotide and said modified nucleotide comprises a2′-modification selected from the group consisting of 2′-aminoethyl,2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 23. The double stranded RNAioligonucleotide (dsRNAi) of claim 22, wherein all nucleotides of theoligonucleotide are modified, and the modification is 2′-fluoro and2′-O-methyl.
 24. The double stranded RNAi oligonucleotide (dsRNAi) ofclaim 22, wherein about 10-15% of the nucleotides of the sense strandcomprise a 2′-fluoro modification; and/or about 25-35% of thenucleotides of the antisense strand comprise a 2′-fluoro modification;and/or about 25-35% of the nucleotides of the oligonucleotide comprise a2′-fluoro modification.
 25. The double stranded RNAi oligonucleotide(dsRNAi) of claim 22, wherein the sense strand comprises 36 nucleotideswith positions 1-36 from 5′ to 3′, wherein positions 8-11 comprise a2′-fluoro modification; the antisense strand comprises 22 nucleotideswith positions 1-22 from 5′ to 3′, and wherein positions 2, 3, 4, 5, 7,10, and 14 comprise a 2′-fluoro modification; and the remainingnucleotides comprise a 2′-O-methyl modification.
 26. The double strandedRNAi oligonucleotide (dsRNAi) of claim 25, wherein (i) theoligonucleotide comprises at least one phosphorothioate linkage, wherein(a) the antisense strand comprises a phosphorothioate linkage betweenpositions 1 and 2, and between positions 2 and 3; or between positions 1and 2, between positions 2 and 3, and between positions 3 and 4, whereinpositions are numbered 1-4 from 5′ to 3′; and/or (b) the antisensestrand is 22 nucleotides in length, and wherein the antisense strandcomprises a phosphorothioate linkage between positions 20 and 21 andbetween positions 21 and 22, wherein positions are numbered 1-22 from 5′to 3′, and/or (ii) the antisense strand comprises a phosphorylatednucleotide at the 5′ terminus, selected from uridine and adenosine,and/or (iii) the 4′-carbon of the sugar of the 5′-nucleotide of theantisense strand comprises a phosphate analog, wherein the phosphateanalog is a 4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy,and/or (iv) the antisense strand comprises an overhang sequence at the3′ terminus, and wherein the overhang sequence is 2 nucleotides inlength selected from the group consisting of AA, GG, AG, and GA.
 27. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 7, wherein the 3′end of the sense strand comprises a stem-loop set forth as S1-L-S2, andwherein (i) S1 is complementary to S2, and S1 and S2 have the samelength and are each 1-10 nucleotides in length; and (ii) L forms a loopbetween S1 and S2 of 3-5 nucleotides in length.
 28. The double strandedRNAi oligonucleotide (dsRNAi) of claim 27, wherein S1 and S2 are 6nucleotides in length; and L is a triloop or a tetraloop.
 29. The doublestranded RNAi oligonucleotide (dsRNAi) of claim 28, wherein L is atetraloop comprising the sequence 5′-GAAA-3′.
 30. The double strandedRNAi oligonucleotide (dsRNAi) of claim 27, wherein the stem-loopcomprises the sequence 5′-GCAGCCGAAAGGCUGC-3′ (SEQ ID NO: 1121).
 31. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 27, wherein atleast one nucleotide of the oligonucleotide is conjugated to one or moretargeting ligands.
 32. The double stranded RNAi oligonucleotide (dsRNAi)of claim 31, wherein each targeting ligand comprises a carbohydrate,amino sugar, cholesterol, polypeptide or lipid.
 33. The double strandedRNAi oligonucleotide (dsRNAi) of claim 31, wherein the targeting ligandis a hepatocyte targeting ligand and each targeting ligand comprises aN-acetylgalactosamine (GalNAc) moiety.
 34. The double stranded RNAioligonucleotide (dsRNAi) of claim 33, wherein the N-acetylgalactosamine(GalNAc) moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety,a trivalent GalNAc moiety or a tetravalent GalNAc moiety.
 35. The doublestranded RNAi oligonucleotide (dsRNAi) of claim 31, wherein the stemloop comprises one or more targeting ligands conjugated to one or morenucleotides of the stem loop or the loop comprises one or more targetingligands conjugated to one or more nucleotides of the loop.
 36. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 35, wherein theone or more targeting ligands is conjugated to one or more nucleotidesof the loop, wherein the loop comprises 4 nucleotides numbered 1-4 from5′ to 3′, wherein nucleotides at positions 2, 3, and 4 each comprise oneor more targeting ligands, and wherein the targeting ligands are thesame or different.
 37. The double stranded RNAi oligonucleotide (dsRNAi)of claim 35, wherein the targeting ligand is a hepatocyte targetingligand and each targeting ligand comprises a N-acetylgalactosamine(GalNAc) moiety.
 38. The double stranded RNAi oligonucleotide (dsRNAi)of claim 37, wherein the N-acetylgalactosamine (GalNAc) moiety is amonovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAcmoiety or a tetravalent GalNAc moiety.
 39. The double stranded RNAioligonucleotide (dsRNAi) of claim 35, wherein the targeting ligand is ahepatocyte targeting ligand and up to 4 nucleotides of L of thestem-loop are each conjugated to a monovalent GalNAc moiety.
 40. Thedouble stranded RNAi oligonucleotide (dsRNAi) of claim 27, comprising atleast one modified nucleotide and said modified nucleotide comprises a2′-modification selected from the group consisting of 2′-aminoethyl,2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 41. The double stranded RNAioligonucleotide (dsRNAi) of claim 40, wherein all nucleotides of theoligonucleotide are modified, and the modification is 2′-fluoro and2′-O-methyl.
 42. The double stranded RNAi oligonucleotide (dsRNAi) ofclaim 40, wherein about 10-15%, of the nucleotides of the sense strandcomprise a 2′-fluoro modification; and/or about 25-35% of thenucleotides of the antisense strand comprise a 2′-fluoro modification;and/or about 25-35% of the nucleotides of the oligonucleotide comprise a2′-fluoro modification.
 43. The double stranded RNAi oligonucleotide(dsRNAi) of claim 40, wherein the sense strand comprises 36 nucleotideswith positions 1-36 from 5′ to 3′, wherein positions 8-11 comprise a2′-fluoro modification; the antisense strand comprises 22 nucleotideswith positions 1-22 from 5′ to 3′, and wherein positions 2, 3, 4, 5, 7,10, and 14 comprise a 2′-fluoro modification; and the remainingnucleotides comprise a 2′-O-methyl modification.
 44. The double strandedRNAi oligonucleotide (dsRNAi) of claim 43, wherein (i) theoligonucleotide comprises at least one phosphorothioate linkage, wherein(a) the antisense strand comprises a phosphorothioate linkage betweenpositions 1 and 2, and between positions 2 and 3; or between positions 1and 2, between positions 2 and 3, and between positions 3 and 4, whereinpositions are numbered 1-4 from 5′ to 3′; and/or (b) the antisensestrand is 22 nucleotides in length, and wherein the antisense strandcomprises a phosphorothioate linkage between positions 20 and 21 andbetween positions 21 and 22, wherein positions are numbered 1-22 from 5′to 3′, and/or (ii) the antisense strand comprises a phosphorylatednucleotide at the 5′ terminus, selected from uridine and adenosine,and/or (iii) the 4′-carbon of the sugar of the 5′-nucleotide of theantisense strand comprises a phosphate analog, wherein the phosphateanalog is a 4′-phosphate analog comprising 5′-methoxyphosphonate-4′-oxy,and/or (iv) the antisense strand comprises an overhang sequence at the3′ terminus, and wherein the overhang sequence is 2 nucleotides inlength selected from the group consisting of AA, GG, AG, and GA.
 45. Apharmaceutical composition comprising a double stranded RNAioligonucleotide of claim 1, and a pharmaceutically acceptable carrier,delivery agent or excipient.
 46. A method of treating non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),multiple sclerosis, or systemic lupus erythematosus, comprisingadministering to a patient in need thereof a double stranded RNAioligonucleotide of claim
 1. 47. A method of treating non-alcoholic fattyliver disease (NAFLD), non-alcoholic steatohepatitis (NASH), multiplesclerosis, or systemic lupus erythematosus, comprising administering toa patient in need thereof the pharmaceutical composition of claim 45.